Biology subjects

Naganathan, A. N.

Publications and source records attributed to Naganathan, A. N..

11 recordsLinked to original sources

bioRxiv2026

Switching Functional DNA-Binding Modes by Tuning Protein Order-Disorder Equilibria

Physical remodeling of chromatin by non-histone architectural proteins of the High Mobility Group B (HMGB) family is central to eukaryotic transcriptional regulation. Nhp6A, the prototypical single-HMG-box protein from yeast, harbors both ordered and disordered regions enabling it to bind and bend DNA without sequence specificity. Here, we integrate ensemble experiments, single-molecule FRET, statistical mechanical modeling and atomistic simulations to dissect the structural and functional consequences of context-dependent phosphorylation in the ordered domain and its interplay with the intrinsically disordered region in Nhp6A. We find that Nhp6A occupies a narrow thermodynamic window, with a melting temperature close to the growth temperature of its host organism and high unfolding cooperativity, a feature conserved across the HMG-box family. Phosphorylation extents - mimicked by multisite phosphomimetic substitutions at residue positions conserved across fungal taxa - smoothly tuning the conformational equilibria between at least two different substates in the native ensemble, apart from the unfolded state. This intrinsic plasticity enables close packing of Nhp6A on DNA through two degenerate binding modes, accompanied by two distinct DNA bending geometries. DNA rescues a strongly destabilized mutant, T63D, through favorable intermolecular interactions, thus effectively acting as a chaperone driving folding. Our findings thus reveal a conserved sequence-ensemble-dynamics code in Nhp6A wherein not just stability, but also phosphorylation-induced conformational switching, disordered tail dynamics, and DNA binding-bending closely coordinate chromatin accessibility. The combination of marginal stability, large cooperativity and electrostatic frustration emerges as a design principle to encode charge sensitivity into proteins, and may represent a general strategy for multisite post-translational regulation.

biophysics↗

bioRxiv2026

Hashi: Bridging Statistical Model Derived 1D Microstate Encodings and Protein 3D Structural Ensembles

The functioning of proteins is intimately linked to the conformational states they sample within the native ensemble. Generating ensembles from a single static structure is therefore a research domain receiving considerable attention. In this application note, we introduce Hashi, a pipeline to rapidly generate realistic structural ensembles from the outputs of the structure-based Wako-Saito-Munoz-Eaton (WSME) statistical mechanical model of protein folding. This approach relies on integrating the block WSME model outputs - strings of zeros and ones describing the conformational status of every residue over thousands or millions of microstates each assigned a statistical weight derived from physically grounded energy-entropy terms, and free energy profiles - with the RANCH module of the EOM (ensemble optimization method) from the ATSAS software suite, providing three-dimensional views of the structural ensembles within the model framework. It is applicable to a variety of single-chain monomeric systems with lengths ranging from 30 to 500 residues, including globular and repeat proteins. Ensembles can be generated within seconds to minutes on a standard computer, without recourse to high-performance computational clusters. The generated structural ensembles can also be rank-ordered according to their free energies within a given macrostate or a range of reaction coordinate values. Since the statistical weights of the WSME model microstates can be reweighted or calibrated with experiments, the ensembles shed light on not just the folding mechanism but also on the structural excursions that determine function and opening of otherwise buried binding pockets.

biophysics↗

bioRxiv2026

A Structural Domain in the genomic RNA of SARS-CoV-2 Folds into a Compact Granular Structure without the N protein: A Single-Molecule Fluorescence Spectroscopic Investigation

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) packages its single-stranded genomic RNA (gRNA) having about 30,000 nucleotides into virions by forming 35-40 granular ribonucleoprotein (RNP) units. Each RNP unit has a diameter of [~]15 nm. While it is generally assumed that the assembly of these RNPs is driven by the binding of the nucleocapsid (N) protein to the gRNA in the cytoplasm, the precise molecular mechanism remains to be fully elucidated. In this study, we develop an experimental strategy based on single-molecule fluorescence and fluorescence correlation spectroscopies to examine the formation of long-range base pairing within a candidate structural domain corresponding to nt 12230-12686 of the gRNA (gRNA12k). Our results demonstrate that the 5 and 3 regions of gRNA12k autonomously form long-range base pairing in near-physiological buffers containing mono- and divalent cations, independently of the N protein. This domain possesses an extensive secondary structure, is compact, and can unfold and refold reversibly upon heat treatment and cooling. Notably, the addition of the N protein melts the long-range base pairs, and causes the aggregation of multiple molecules of gRNA12k. Based on these observations, we propose a refined mechanism for the genome assembly in SARS-CoV-2: gRNA initially forms autonomous granular structures, which are subsequently reorganized and condensed by the N protein to chaperone the assembly of the entire gRNA. SignificanceSARS-CoV-2 organizes its exceptionally long genomic RNA (gRNA) having about 30,000 nt into 35-40 granular ribonucleoprotein (RNP) units for viral packaging. It has been assumed that the nucleocapsid (N) protein drives the formation of the RNP granules. In this study, we challenge this prevailing view by demonstrating that a specific region of the gRNA sequence inherently encodes the information to fold into a compact, granular architecture independently of any proteins. Unexpectedly, we found that the N protein partially melts the autonomous structures, suggesting that it acts as an RNA chaperone to facilitate flexible genome assembly. Our findings redefine the interplay between viral proteins and gRNA, offering a new perspective on the mechanism of coronavirus replication.

biophysics↗

bioRxiv2025

Diverse Conformational Ensembles Define the Shared Folding-Allosteric Landscapes of Protein Kinases

Sequence variation across members of an enzyme family contributes to diverse ensemble behaviors, which subtly influence substrate affinity, selectivity and regulation. A classic example is the family of eukaryotic protein kinases (EPKs), which regulate numerous cellular processes and serve as important drug targets. Here, we dissect the consequences of sequence variation on the folding-conformational landscapes by performing a meta-analysis of 274 EPKs through a structure-based statistical mechanical framework. We find that EPKs populate several partially structured states in their native ensemble with a hierarchy of structural order in the N-terminal lobe that is critical for catalysis and activation. Despite this, the unfolding mechanism is uniquely conserved across the majority of kinases, with the N-terminal lobe unfolding first. Kinase activation modulates the local stability and thermodynamic connectivity in a non-conserved manner and across the entire structure, due to the strong coupling between the active site residues to distant sites, including the established allosteric pockets. We further show how activation drives the Abl kinase ensemble towards a more folded and thermodynamically coupled system in a graded manner. Our work uncovers the thermodynamic design principles of kinases with insights into allostery, while shedding light on the extents to which ensemble behaviors are impacted by sequence variations in paralogs.

biophysics↗

bioRxiv2024

Polyphosphate Discriminates Protein Conformational Ensembles More Efficiently than DNA Promoting Diverse Assembly and Maturation Behaviors

Disordered proteins and domains often assemble into condensates with polyanionic nucleic acids, primarily via charge complementarity, regulating numerous cellular functions. However, the assembly mechanisms associated with the other abundant and ubiquitous, anionic, stress-response regulating polymer, polyphosphate (polyP), is less understood. Here, we employ the intrinsically disordered DNA binding domain (DBD) of cytidine repressor (CytR) from E.coli to study the nature of assembly processes with polyP and DNA. CytR forms metastable liquid-like condensates with polyP and DNA, while undergoing liquid-to-solid transition in the former and dissolving in the latter. On mutationally engineering the ensemble to exhibit more or less structure and dimensions than the WT, the assembly process with polyP is directed to either condensates with partial time-dependent dissolution or spontaneous aggregation, respectively. On the other hand, the CytR variants form only liquid-like but metastable droplets with DNA which dissolve within a few hours. Polyphosphate induces large secondary-structure changes, with two of the mutants adopting polyproline II-like structures within droplets, while DNA has only minimal structural effects. Our findings reveal how polyphosphate can more efficiently discern conformational heterogeneity in the starting protein ensemble, its structure, and compactness, with broad implications in assembly mechanisms involving polyP and stress response in bacterial systems.

biophysics↗

bioRxiv2024

The lack of trade-off between conformational stability and binding affinity in a nanobody with therapeutic potential for a misfolding disease

To improve protein pharmaceuticals, we need to balance protein stability and binding affinity with in vivo efficiency. We have recently developed a nanobody (NB-AGT-2) against the alanine:glyoxylate aminotransferase with high stability (Tm[~]85{degrees}C) that may be useful to treat a misfolding disease called primary hyperoxaluria type 1. In this work, we characterize the relationships between protein stability and binding affinity in NB-AGT-2 by generating single and double cavity-creating mutants in its hydrophobic core. These mutations decrease thermal stability by 10-20 {degrees}C, reflecting changes in thermodynamic stability of up to 8 kcal{middle dot}mol-1, hardly affecting their binding affinity for its target. Our results thus show that NB stability can be challenged without an effect on its binding.

biophysics↗

bioRxiv2024

Oligomerization-Mediated Phase-Separation in the Nucleoid-Associated Sensory Protein H-NS is Controlled by Ambient Cues

H-NS, a nucleoid-associated protein (NAP) from enterobacteria, regulates gene expression by dynamically transducing environmental cues to conformational assembly and DNA binding. In this work, we show that H-NS from Escherichia coli, which can assemble into octameric and tetrameric oligomerization states, forms spontaneous micron-sized liquid-like condensates with DNA at sub-physiological concentrations in vitro. The heterotypic condensates are metastable at 298 K, partially solubilizing with time, while still retaining their liquid-like properties. The condensates display UCST-like phase behavior solubilizing at higher temperatures, but with a large decrease in droplet-assembly propensities at 310 K and also at higher ionic strength. Condensate formation can be tuned in a cyclic manner between 298 and 310 K with the extent of reversibility determined by the incubation time, highlighting strong hysteresis. An engineered phospho-mimetic variant (Y61E) of H-NS, which is dimeric and only weakly binds DNA, is unable to form condensates. The Y61E mutant solubilizes pre-formed H-NS condensates with DNA in a few minutes with nearly an order of magnitude speed-up in droplet dissolution at 310 K relative to 298 K, demonstrating rapid molecular transport between dilute and condensed phases. Our results establish that the oligomerization of H-NS is intrinsically tied not only to DNA binding but also its phase-separation tendencies, while showcasing the regulatable and programmable nature of heterotypic condensates formed by an archetypal NAP via multiple cues and their lifetimes.

biophysics↗

bioRxiv2023

Conformational Tuning Shapes the Balance between Functional Promiscuity and Specialization in Paralogous Plasmodium Acyl-CoA Binding Proteins

Paralogous proteins confer enhanced fitness to organisms via complex sequence-conformation codes that shape functional divergence, specialization, or promiscuity. Here, we resolve the underlying mechanism of promiscuous binding versus partial sub-functionalization in paralogs by studying structurally-identical Acyl-CoA Binding Proteins (ACBPs) from Plasmodium falciparum that serve as promising drug targets due to their high expression during the protozoan proliferative phase. Combining spectroscopic measurements, solution NMR, SPR and simulations on two of the paralogs, A16 and A749, we show that minor sequence differences shape nearly every local and global conformational feature. A749 displays a broader and heterogeneous native ensemble, weaker thermodynamic coupling and cooperativity, enhanced fluctuations, and a larger binding-pocket volume, compared to A16. Site-specific tryptophan probes signal a graded reduction in the sampling of substates in the holo form, which is particularly more apparent in A749, hinting at conformational-selection-like mechanism of binding. The paralogs exhibit a spectrum of binding affinities to different acyl-CoAs with A749, the more promiscuous and hence the likely ancestor, binding 1000-fold stronger to Lauroyl-CoA under physiological conditions. We thus demonstrate how minor sequence changes modulate the extent of long-range interactions and dynamics, effectively contributing to the molecular evolution of contrasting functional repertoires in paralogs.

biophysics↗

bioRxiv2022

Thermodynamic Architecture and Conformational Plasticity of GPCRs

G-protein-coupled receptors (GPCRs) are ubiquitous integral membrane proteins involved in diverse cellular signaling processes and consequently serve as crucial drug targets. Here, we carry out the first large-scale ensemble thermodynamic study of 45 different ligand-free GPCRs employing a structure-based statistical mechanical framework and identify extensive conformational plasticity encompassing the seven transmembrane (TM) helices. Multiple partially structured states or intermediates co-exist in equilibrium in the native ensemble, with the TM helices 1, 6 and 7 displaying varied degrees of structure, and TM3 exhibiting the maximal stability. Active state GPCRs are characterized by reduced conformational heterogeneity with altered coupling-patterns distributed not just locally but throughout the structural scaffold. Strongly coupled residues are distributed across the structure in an anisotropic manner accounting for only 13% of the residues, highlighting that a large number of residues in GPCRs are inherently dynamic to enable structural motions critical for function. Our work thus uncovers the thermodynamic hallmarks of GPCR structure and activation, and how differences quantifiable only via higher-order coupling free energies provide insights into their exquisite structural specialization and the fluid nature of the intramolecular interaction network. The intricate landscapes and perturbation methodologies presented here lay the foundation for understanding allosteric mechanisms in GPCRs, location of structural-functional hot-spots, and effects of disease-causing mutations.

biophysics↗

bioRxiv2021

Combining ancestral reconstruction with folding-landscape simulations to engineer heterologous protein expression

Obligate symbionts exhibit high evolutionary rates and extensive sequence divergence. Here, we use the thioredoxin from Candidatus Photodesmus katoptron, an uncultured symbiont of flashlight fish, to explore evolutionary and engineering aspects of protein folding in heterologous hosts. The symbiont protein is a standard thioredoxin in terms of 3D-structure, stability and redox activity. However, its refolding in vitro is very slow and its expression in E. coli leads to insoluble protein. By contrast, resurrected Precambrian thioredoxins express efficiently in E. coli, plausibly reflecting an ancient adaptation to unassisted folding. We have used a statistical-mechanical model of the folding landscape to guide back-to-ancestor engineering of the symbiont protein. Remarkably, we find that the efficiency of heterologous expression correlates with the in vitro refolding rate and that the ancestral expression efficiency can be achieved with only 1-2 back-to-ancestor replacements. These results demonstrate a sequence-engineering approach to rescue inefficient heterologous expression, a major biotechnological bottleneck.

biophysics↗

bioRxiv2020

The metal cofactor zinc and interacting membranes modulate SOD1 conformation-aggregation landscape in an in vitro ALS Model

Aggregation of Cu-Zn superoxide dismutase (SOD1) is implicated in the motor neuron disease, ALS. Although more than 140 disease mutations of SOD1 are available, their stability or aggregation behaviors in membrane environment are not correlated with disease pathophysiology. Here, we use multiple mutational variants of SOD1 to show that the absence of Zn, and not Cu, significantly impacts membrane attachment of SOD1 through two loop regions facilitating aggregation driven by lipid induced conformational changes. These loop regions influence both the primary (through Cu intake) and the gain of function (through aggregation) of SOD1 presumably through a shared conformational landscape. Combining experimental and theoretical frameworks using representative ALS disease mutants, we develop a co-factor derived membrane association model wherein mutational stress closer to the Zn (but not to the Cu) pocket is responsible for membrane association mediated toxic aggregation and survival time scale after ALS diagnosis.

biophysics↗