Biology subjects

Athreya, A.

Publications and source records attributed to Athreya, A..

8 recordsLinked to original sources

bioRxiv2026

Structural basis of K+/H+ antiport in YcgO and its inhibition by unphosphorylated PtsN

Cation-proton antiporters (CPAs) are vital for the maintenance of ionic homeostasis and normal physiology among diverse cell types. Despite recent insights into K+/H+ exchange transporters, the diversity in their structural organization and regulatory mechanisms of K+-specific CPAs are minimally understood. Here, we explore the architecture of an E. coli CPA1 K+/H+ antiporter, YcgO and its inhibition by the unphosphorylated form of PtsN, the terminal protein of a regulatory phosphorelay, using cryoEM structures at 3.4 [A] and 3.2 [A] resolution, respectively. Homodimeric YcgO bound to K+ ions in the occluded conformation, harbors additional linked cytosolic domains, RCK and CorC, to regulate the movement of the transport helices within the YcgO dimer. These domains are the sites of interaction and efflux inhibition by unphosphorylated PtsN, which interacts with the CorC domains with high affinity and allosterically augments inhibitory interactions of CorC with transport helices of YcgO. Inhibition is relieved leading to constitutive activation, upon disrupting the CorC-transport conduit interface. This study illuminates the structural basis of K+ efflux mediated through regulation of a K+/H+ antiporter in E. coli and related prokaryotes via a metabolic network involving a regulatory phosphorelay.

biochemistry↗

bioRxiv2026

Valency-Limited Molecular Dynamics Simulations of Stickers-and-Spacers Polymers Reveal a Tradeoff Between Condensation and Organization

Biomolecular condensates formed by intrinsically disordered proteins (IDPs) are often described using stickers-and-spacers models, in which specific sticker motifs form reversible crosslinks and spacer regions modulate phase behavior. Recent theory predicts that heterogeneous nonspecific spacer interactions can promote condensation but may also disrupt sticker-mediated organization. Here, we develop an off-lattice coarse-grained stickers-and-spacers polymer model for continuous molecular dynamics simulations and implement it in the GPU-accelerated OpenABC package. The model uses a directional sticker-sticker interaction to encode limited valency through interaction geometry, producing effectively one-to-one sticker binding without explicit bond assignment. Simulations of one-component systems show that sticker affinity and multivalency promote porous, network-like condensates, while nonspecific spacer interactions can also drive phase separation but produce more compact, spacer-dominated dense phases. When both interaction types are present, strong spacer heterogeneity reduces sticker conversion, suppresses sticker mobility, and disrupts the sticker-mediated network. In two-component systems, specific sticker interactions buffer client recruitment into host condensates, while nonspecific spacer interactions produce reservoir-dependent uptake. These results support a tradeoff in which spacer heterogeneity promotes condensation at the cost of condensate organization and compositional robustness, providing a physical rationale for the suppression of promiscuous spacer interactions in low-complexity IDP regions.

biophysics↗

bioRxiv2024

Gut Microbiome Signatures During Acute Infection Predict Long COVID

Long COVID (LC), manifests in 10-30% of non-hospitalized individuals post-SARS-CoV-2 infection leading to significant morbidity. The predictive role of gut microbiome composition during acute infection in the development of LC is not well understood, partly due to the heterogeneous nature of disease. We conducted a longitudinal study of 799 outpatients tested for SARS-CoV-2 (380 positive, 419 negative) and found that individuals who later developed LC harbored distinct gut microbiome compositions during acute infection, compared with both SARS-CoV-2-positive individuals who did not develop LC and negative controls with similar symptomatology. However, the temporal changes in gut microbiome composition between the infectious (0-1 month) and post-infectious (1-2 months) phases was not different between study groups. Using machine learning, we showed that microbiome composition alone more accurately predicted LC than clinical variables. Including clinical data only marginally enhanced this prediction, suggesting that microbiome profiles during acute infection may reflect underlying health status and immune responses thus, help predicting individuals at risk for LC. Finally, we identified four LC symptom clusters, with gastrointestinal and fatigue-only groups most strongly linked to gut microbiome alterations.

genomics↗

bioRxiv2024

Assessing the impact of Brd2 depletion on chromatin compartmentalization

Recent insights into genome organization have emphasized the importance of A/B chromatin compartments. While our previous research showed that Brd2 depletion weakens compartment boundaries and promotes A/B mixing 1, Hinojosa-Gonzalez et al.2 were unable to replicate the findings. In response, we revisited our Micro-C data and successfully replicated the original results using the default parameters in the cooltools software package. We show that, after correcting inconsistencies with the selection and phasing of the compartment profiles, the decrease in B compartment strength persists but the change in compartment identity is to a much lesser extent than originally reported. To further assess the regulatory role of Brd2, we used saddle plots to determine the strength of compartmentalization and observed a consistent decrease of compartment strength especially at B compartments upon Brd2 depletion. This study highlights the importance of selecting appropriate parameters and analytical tools for compartment analysis and carefully interpreting the results.

genomics↗

bioRxiv2023

Electrostatic encoding of genome organization principles within single native nucleosomes

The eukaryotic genome, first packed into nucleosomes of about 150 bp around the histone core, is organized into euchromatin and heterochromatin, corresponding to the A and B compartments, respectively. Here, we asked if individual nucleosomes in vivo know where to go. That is, do mono-nucleosomes by themselves contain A/B compartment information, associated with transcription activity, in their biophysical properties? We purified native mono-nucleosomes to high monodispersity and used physiological concentrations of biological polyamines to determine their condensability. The chromosomal regions known to partition into A compartments have low condensability and vice versa. In silico chromatin polymer simulations using condensability as the only input showed that biophysical information needed to form compartments is all contained in single native nucleosomes and no other factors are needed. Condensability is also strongly anticorrelated with gene expression, and especially so near the promoter region and in a cell type dependent manner. Therefore, individual nucleosomes in the promoter know whether the gene is on or off, and that information is contained in their biophysical properties. Comparison with genetic and epigenetic features suggest that nucleosome condensability is a very meaningful axis onto which to project the high dimensional cellular chromatin state. Analysis of condensability using various condensing agents including those that are protein-based suggests that genome organization principle encoded into individual nucleosomes is electrostatic in nature. Polyamine depletion in mouse T cells, by either knocking out ornithine decarboxylase (ODC) or inhibiting ODC, results in hyperpolarized condensability, suggesting that when cells cannot rely on polyamines to translate biophysical properties of nucleosomes to control gene expression and 3D genome organization, they accentuate condensability contrast, which may explain dysfunction known to occur with polyamine deficiency.

biophysics↗

bioRxiv2022

CryoEM structure of QacA, an antibacterial efflux transporter from Staphylococcus aureus

Efflux of antibacterial compounds is a major mechanism for developing antimicrobial resistance. In the Gram-positive pathogen Staphylococcus aureus, QacA, a 14 transmembrane (TM) helix containing major facilitator superfamily antiporter, mediates proton-coupled efflux of mono and divalent cationic antibacterial compounds. In this study, we report the cryoEM structure of QacA, with a single mutation D411N that improves homogeneity and retains efflux activity against divalent cationic compounds like dequalinium and chlorhexidine. The structure of substrate-free QacA, complexed to two single-domain camelid antibodies, was elucidated to a resolution of 3.6 [A]. The structure displays an outward-open conformation with an extracellular hairpin loop, which is conserved in a subset of DHA2 transporters and its deletion causes a loss of function in the transporter. Modeling and simulations of QacAs cytosol-facing and occluded conformations reveal asymmetry in the rocker-switch mode of QacAs conformational shifts, providing new insights into the organization and structural dynamics of DHA2 members.

biochemistry↗

bioRxiv2020

Structural basis of inhibition of a putative drug efflux transporter NorC, through a single-domain camelid antibody

Multi-drug efflux is a major mechanism of acquiring antimicrobial resistance among superbugs. In this study, we report the X-ray structure of NorC, a 14 transmembrane major facilitator superfamily member that is implicated in fluoroquinolone resistance in drug-resistant Staphylococcus aureus strains, at a resolution of 3.6 [A]. The NorC structure was determined in complex with a single-domain camelid antibody that interacts at the extracellular face of the transporter and stabilizes it in an outward-open conformation. The complementarity determining regions of the antibody enter and block solvent access to the interior of the vestibule, thereby inhibiting alternating-access. NorC specifically interacts with an organic cation, tetraphenylphosphonium, although it does not demonstrate an ability to transport it. The interaction is compromised in the presence of NorC-antibody complex, consequently establishing a strategy to detect and block NorC and related efflux pumps through the use of single- domain camelid antibodies.

biochemistry↗

bioRxiv2018

Dissection of protonation sites for antibacterial recognition and transport in QacA, a multi-drug efflux transporter.

QacA is a drug:H+ antiporter (DHA2) with 14 transmembrane helices, that renders antibacterial resistance to methicillin-resistant Staphylococcus aureus (MRSA) strains, with homologues in other pathogenic organisms. It is a highly promiscuous antiporter, capable of H+- driven efflux of a wide array of cationic antibacterial compounds and dyes. Our study, using a homology model of QacA, reveals a group of six protonatable residues in its vestibule. Systematic mutagenesis resulted in identification of D34 (TM1), and a cluster of acidic residues in TM13 including E407 and D411 and D323 in TM10, as being crucial for substrate recognition and transport of monovalent and divalent cationic antibacterial compounds. The transport and binding properties of QacA and its mutants were explored using whole cells, inside-out vesicles, substrate-induced H+ release and microscale thermophoresis. We identify two sites, D34 and D411 as vital players in substrate recognition while E407 facilitates substrate efflux as a protonation site. We also observe that E407 plays a moonlighting role as a substrate recognition site for dequalinium transport. These observations rationalize the promiscuity of QacA for diverse substrates. The study unravels the role of acidic residues in QacA with implications for substrate recognition, promiscuity and processive transport in multidrug efflux transporters, related to QacA.\n\nHighlightsO_LIA homology model of QacA with 14 TM helices was used to test the importance of acidic residues within the vestibule.\nC_LIO_LIMono and Divalent cationic substrate recognition requires two sites D34 (TM1) and D411 (TM13).\nC_LIO_LIE407 (TM13) is important for protonation driven efflux.\nC_LIO_LISubstrate recognition of a divalent substrate, dequalinium occurs at E407 instead of D411 providing glimpses into the promiscuity of substrate recognition in QacA.\nC_LI

biochemistry↗