Biology subjects

Dastvan, R.

Publications and source records attributed to Dastvan, R..

8 recordsLinked to original sources

bioRxiv2026

Protonation- and substrate-regulated dimer opening couples brain-type creatine kinase to vesicular and actin-remodeling membranes

Brain-type creatine kinase (CK-BB) buffers local ATP demand through reversible phosphotransfer between ATP and phosphocreatine, yet how this soluble metabolic enzyme engages membrane compartments is unknown. Here, we combine fluorescence microscopy, DEER spectroscopy, hydrogen-deuterium exchange and native mass spectrometry, DEER- and AlphaFold-guided modeling, and long-timescale molecular dynamics to define the pH- and substrate-regulated conformational landscape governing CK-BB membrane association. Acidification promotes curvature-sensitive membrane binding and redistributes endogenous and recombinant CK-BB from diffuse cytosolic pools to punctate vesicular structures and membrane ruffles. Substrates independently promote curvature-sensitive association at neutral pH. DEER and modeling reveal an asymmetric dimer in which the convex surface remains restrained, whereas the concave catalytic-regulatory surface samples pH- and substrate-dependent intermediates. We identify progressive dimer opening as a novel regulatory mechanism whereby acidification and substrate binding increase dynamics across the convex surface and N-terminal dimer interface, generating membrane-competent conformations that facilitate curvature sensing and membrane association. Substrate binding buffers acid-induced deprotection while preserving dynamics near the His191/Ser199 regulatory interface. These findings establish CK-BB as a previously unrecognized curvature-sensitive metabolic enzyme and define dimer dynamics as a molecular switch coupling protonation and substrate occupancy to curved-membrane recognition and localized ATP regeneration, with potential relevance to neurodegeneration and cellular stress.

biophysics↗

bioRxiv2026

Proton-coupled alternating access in a versatile Spns drug efflux pump from Mycobacterium smegmatis

Spns transporters are a mechanistically distinct branch of the major facilitator superfamily that regulate lipid transport, lysosomal homeostasis, immunity and disease. How the conserved Spns fold integrates protonation, substrate binding and alternating access to support chemically and directionally diverse transport remains unresolved. Here we combine DEER spectroscopy in lipid nanodiscs with DEER-and AlphaFold-guided modeling and protonation-mimetic mutagenesis to define the conformational landscape of the Mycobacterium smegmatis homolog MsSpns. Protonation shifts MsSpns toward an inward-facing state, whereas deprotonation favors a broader outward-facing ensemble through remodeling of intracellular and extracellular gates. Protonation-mimetic substitutions identify Glu126 as a switch that stabilizes an inward-facing, substrate-entry-competent conformation, while Asp38 and Asp57 favor outward-facing states and tune the extracellular proton-sensing network. The substrate-binding cavity displays distinct proton sensitivity and weaker cooperativity than gating networks. Hydrophilic cationic substrates stabilize the outward-facing state, consistent with efflux antiport, whereas lipophilic compounds favor the inward-facing state, suggesting uptake or allosteric stabilization. Thus, conserved proton-coupling elements can power opposing transport modes, revealing the mechanistic versatility of the Spns fold and its therapeutic potential.

biophysics↗

bioRxiv2026

A Phosphorylation Switch Modulates Configurational Codes in the Oncofetal IGF2BP RNA Binding Paralogs

The insulin like growth factor 2 mRNA binding proteins (IGF2BP1-3) are oncofetal RNA regulators that control translation, stability, and localization of several transcripts, yet display paralog specific functions despite high structural similarity. Each paralog contains six RNA binding domains (two RRMs and four KH domains) linked by intrinsically disordered segments. mTORC2 phosphorylates IGF2BP1 and IGF2BP3 at a single conserved serine within the disordered linker between the RRM2 and KH1 domains, a modification required for proper regulation of mRNA translational fate. Pairing site specific phosphoserine incorporation with structural and biophysical interrogations, we show that this phosphorylation acts as a configurational switch that reorganizes long range arrangements of RNA binding domains and linkers without altering the secondary structure, and with only modest effects on RNA binding affinity. Critically, pSer driven rearrangements occur both in the RNA free state and upon RNA engagement, and the resulting architectures differ markedly between IGF2BP1 and IGF2BP3 despite >70% sequence identity. These paralog specific, phosphorylation dependent configurational landscapes likely underlie differences in mRNA recognition modes and functional outcomes. Our work identifies a post translational mechanism that tunes IGF2BP paralog dynamics across free and RNA bound states to program target mRNA selection, processing, and translational fate.

biochemistry↗

bioRxiv2025

Structural dynamics of sphingosine kinase 1 regulation and inhibition

Sphingosine kinase 1 (SK1) generates sphingosine-1-phosphate, a bioactive lipid implicated in cancer and other diseases. Despite its clinical importance, the structural and dynamic basis of SK1 regulation and inhibition remains poorly understood. Using integrated spectroscopic and computational approaches, we uncover conformational transitions that govern substrate entry, catalysis, and inhibitor binding. Phosphorylation of Ser225 reconfigures the regulatory loop and reshuffles salt bridges, priming SK1 for membrane engagement and catalytic activity. We identify a previously uncharacterized catalytic intermediate with a distinct conformation and a highly dynamic lipid-binding loop 1 (LBL-1), sensitive to potent inhibitors such as PF-543. These inhibitors not only stabilize non-catalytic states but also induce LBL-mediated dimerization, blocking membrane binding and substrate access. Our findings reveal a multilayered regulatory mechanism driven by structural flexibility and establish a novel inhibitory paradigm. This framework provides critical insight into SK1 regulation and a foundation for developing next-generation SK1-targeted therapeutics.

biophysics↗

bioRxiv2024

Computational structural prediction and chemical inhibition of the human mitochondrial pyruvate carrier protein heterodimer complex

The mitochondrial pyruvate carrier (MPC) plays a role in numerous diseases including neurodegeneration, metabolically dependent cancers, and the development of insulin resistance. Several previous studies in genetic mouse models or with existing inhibitors suggest that inhibition of the MPC could be used as a viable therapeutic strategy in these diseases. However, the MPCs structure is unknown, making it difficult to screen for and develop therapeutically viable inhibitors. Currently known MPC inhibitors would make for poor drugs due to their poor pharmacokinetic properties, or in the case of the thiazolidinediones (TZDs), off-target specificity for peroxisome-proliferator activated receptor gamma (PPAR{gamma}) leads to unwanted side effects. In this study, we develop several structural models for the MPC heterodimer complex and investigate the chemical interactions required for the binding of these known inhibitors to MPC and PPAR{gamma}. Based on these models, the MPC most likely takes on outward-facing (OF) and inward-facing (IF) conformations during pyruvate transport, and inhibitors likely plug the carrier to inhibit pyruvate transport. Although some chemical interactions are similar between MPC and PPAR{gamma} binding, there is likely enough difference to reduce PPAR{gamma} specificity for future development of novel, more specific MPC inhibitors.

biochemistry↗

bioRxiv2024

Wrapping of single-stranded DNA by Replication Protein A and modulation through phosphorylation

Single-stranded DNA (ssDNA) intermediates, which emerge during DNA metabolic processes are shielded by Replication Protein A (RPA). RPA binds to ssDNA and acts as a gatekeeper, directing the ssDNA towards downstream DNA metabolic pathways with exceptional specificity. Understanding the mechanistic basis for such RPA-dependent specificity requires a comprehensive understanding of the structural conformation of ssDNA when bound to RPA. Previous studies suggested a stretching of ssDNA by RPA. However, structural investigations uncovered a partial wrapping of ssDNA around RPA. Therefore, to reconcile the models, in this study, we measured the end-to-end distances of free ssDNA and RPA-ssDNA complexes using single-molecule FRET and Double Electron-Electron Resonance (DEER) spectroscopy and found only a small systematic increase in the end-to-end distance of ssDNA upon RPA binding. This change does not align with a linear stretching model but rather supports partial wrapping of ssDNA around the contour of DNA binding domains of RPA. Furthermore, we reveal how phosphorylation at the key Ser-384 site in the RPA70 subunit provides access to the wrapped ssDNA by remodeling the DNA-binding domains. These findings establish a precise structural model for RPA-bound ssDNA, providing valuable insights into how RPA facilitates the remodeling of ssDNA for subsequent downstream processes.

biochemistry↗

bioRxiv2023

The PALB2 DNA binding domain is an intrinsically disordered recombinase.

The Partner and Localizer of BRCA2 (PALB2) is a scaffold protein that links BRCA1 with BRCA2 to initiate homologous recombination (HR). PALB2 interaction with DNA strongly enhances HR efficiency in cells. The PALB2 DNA-binding domain (PALB2-DBD) supports strand exchange, a complex multistep reaction conducted by only a few proteins such as RecA-like recombinases and Rad52. Using bioinformatics analysis, small-angle X-ray scattering, circular dichroism, and electron paramagnetic spectroscopy, we determined that PALB2-DBD is an intrinsically disordered region (IDR) forming compact molten globule-like dimer. IDRs contribute to oligomerization synergistically with the coiled-coil interaction. Using confocal single-molecule FRET we demonstrated that PALB2-DBD compacts single-stranded DNA even in the absence of DNA secondary structures. The compaction is bimodal, oligomerization-dependent, and is driven by IDRs, suggesting a novel strand exchange mechanism. Intrinsically disordered proteins (IDPs) are prevalent in the human proteome. Novel DNA binding properties of PALB2-DBD and the complexity of strand exchange mechanism significantly expands the functional repertoire of IDPs. Multivalent interactions and bioinformatics analysis suggest that PALB2 function is likely to depend on formation of protein-nucleic acids condensates. Similar intrinsically disordered DBDs may use chaperone-like mechanism to aid formation and resolution of DNA and RNA multichain intermediates during DNA replication, repair and recombination.

biophysics↗

bioRxiv2022

Protonation-dependent alternating access of a spinster transporter, an emerging family of broad-specificity efflux pumps

Spinster (Spns) lipid transporters are critical for transporting sphingosine-1-phosphate (S1P) across cellular membranes. In humans, Spns2 functions as the main S1P transporter in endothelial cells, making it a potential drug target for modulating S1P signaling. Here, we employed an integrated approach in lipid membranes to identify unknown conformational states of a bacterial Spns from Hyphomonas neptunium (HnSpns) and to define its proton- and substrate-coupled conformational dynamics. Our systematic study reveals conserved residues critical for protonation steps and their regulation, and how sequential protonation of these proton switches coordinates the conformational transitions in the context of a noncanonical ligand-dependent alternating access. A conserved periplasmic salt bridge (Asp60TM2:Arg289TM7) keeps the transporter in a closed conformation, while proton-dependent conformational dynamics are significantly enhanced on the periplasmic side, providing a pathway for ligand exchange. Furthermore, our resistance assays reveal substrate polyspecificity and HnSpns multidrug resistance (MDR) activity that underscore the previously unknown role of Spns proteins in MDR, beyond their activity in sphingolipid transport and signaling.

biophysics↗