Biology subjects

Varghese, I.

Publications and source records attributed to Varghese, I..

3 recordsLinked to original sources

bioRxiv2026

Exploring the GM-CSF Histidine Triad as a Modulator of Structure, Molecular Motion, and Ligand Binding

Granulocyte macrophage-colony stimulating factor (GM-CSF) is a cytokine that plays a role in immune modulation. Its expression is associated with a multitude of different effects ranging from harmful, as in diseases such as rheumatoid arthritis and multiple sclerosis, to beneficial, as in the case of mitigation of diabetes type I and neutropenia. However, there is a large gap in knowledge explaining how GM-CSF toggles its structure for such physiological and pathological interactions. Our work describes an ongoing attempt to address this gap by focusing on a clustered histidine triad within -helices near the N-terminus, which prior studies have suggested play a role in binding ligands at an acidic pH. While GM-CSF is known to be highly flexible at a more acidic pH, several properties of its histidine triad remain unclear at the physiological pH at which GM-CSF would encounter its binding partners. We describe an effort to characterize the role of the GM-CSF histidines under physiological pH, specifically to determine if these histidines are key to GM-CSF structural integrity, and whether individual histidine residues modulate binding as they do at a lower pH. Our findings reveal that, while the histidine residues have an impact on GM-CSF structure, flexibility, and stability, they alone do not modulate the affinity for ligands at neutral pH. These data provide an initial explanation for the pleiotropic functions and interactions of GM-CSF within a biophysical context. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=82 SRC="FIGDIR/small/700583v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@a6fffcorg.highwire.dtl.DTLVardef@1f00c30org.highwire.dtl.DTLVardef@b04c50org.highwire.dtl.DTLVardef@6224d9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2025

Allosteric Modulation of MIF-2 Structure, Catalysis, and Biological Signaling via Cysteine Residues and a Small Molecule, Ebselen

The macrophage migration inhibitory factor (MIF) family of cytokines comprised of the MIF and D-dopachrome tautomerase (or MIF-2) paralogs share identical tertiary and quaternary structures that contribute to their overlapping enzymatic and signaling activities. Recent investigations of MIF and MIF-2 have shown them to possess N-to-C-terminal allosteric crosstalk, but despite the similarity of this "allosteric pathway," its regulation of MIF and MIF-2 is not identical. Thus, structure alone does not preserve the precise allosteric mechanism and additional residues that modulate MIF and MIF-2 allosteric function must be characterized. Cysteines have been identified as allosteric switches for the same biochemical functions of MIF and small molecules targeting its N-terminal enzymatic site have affected the structure of three proximal cysteines. Ebselen is a compound that forms covalent selenylsulfide bonds with MIF cysteines and is hypothesized to destabilize and dissociate the MIF trimer into monomers. Ebselen-bound MIF also displays little-to-no catalysis or biological signaling. However, it is unclear whether Ebselen similarly affects the MIF-2 paralog, despite MIF-2 containing two related cysteines (MIF contains three). We used mutagenesis, nuclear magnetic resonance (NMR), molecular dynamics (MD) simulations, in vitro and in vivo biochemistry to investigate the mechanism of Ebselen as an allosteric modulator of MIF-2 via its cysteines. Our findings suggest that Ebselen partially disrupts the MIF-2 homotrimer, though the overall population of such a structure is <35%, even on the timescale of many hours. Ebselen does attenuate the biological functions of MIF-2 and solution structural biology captures the conformational transitions preceding the destabilized MIF-2 trimer. Significance StatementThe nearly identical MIF and MIF-2 structures have been recently shown to contain an N-to-C-terminal crosstalk between non-overlapping functional sites in vitro and in vivo. Small molecule inhibitors designed as therapeutics for the MIF superfamily have primarily targeted the N-terminal active site, while very few allosteric molecules have been reported. Ebselen, however, destabilizes MIF by covalently modifying its distal Cys80 residue and disrupting its obligate trimeric assembly. This unique mechanism has never been evaluated in the MIF-2 paralog, and this study reports the structural, dynamic, and functional impact of Ebselen on MIF-2. We define the essential structural hallmarks preceding trimer dissociation and reveal that Ebselen inhibits MIF-2 function before trimer disruption, leading to destabilized monomer-monomer interfaces that slowly degrade the quaternary assembly.

biophysics↗

bioRxiv2025

FastConformation: A Standalone ML-Based Toolkit for Modeling and Analyzing Protein Conformational Ensembles at Scale

Deep learning approaches like AlphaFold 2 (AF2) have revolutionized structural biology by accurately predicting the ground state structures of proteins. Recently, clustering and subsampling techniques that manipulate multiple sequence alignment (MSA) inputs into AlphaFold to generate conformational ensembles of proteins have also been proposed. Although many of these techniques have been made open source, they often require integrating multiple packages and can be challenging for researchers who have a limited programming background to employ. This is especially true when researchers are interested in subsampling to produce predictions of protein conformational ensembles, which require multiple computational steps. This manuscript introduces FastConformation, a Python-based application that integrates MSA generation, structure prediction via AF2, and interactive analysis of protein conformations and their distributions, all in one place. FastConformation is accessible through a user-friendly GUI suitable for non-programmers, allowing users to iteratively refine subsampling parameters based on their analyses to achieve diverse conformational ensembles. Starting from an amino acid sequence, users can make protein conformation predictions and analyze results in just a few hours on their local machines, which is significantly faster than traditional molecular dynamics (MD) simulations. Uniquely, by leveraging the subsampling of MSAs, our tool enables the generation of alternative protein conformations. We demonstrate the utility of FastConformation on proteins including the Abl1 kinase, LAT1 transporter, and CCR5 receptor, showcasing its ability to predict and analyze the protein conformational ensembles and effects of mutations on a variety of proteins. This tool enables a wide range of high-throughput applications in protein biochemistry, drug discovery, and protein engineering.

biophysics↗