Biology subjects

Torabifard, H.

Publications and source records attributed to Torabifard, H..

11 recordsLinked to original sources

bioRxiv2026

Small Structural Variations, Large Functional Consequences: Comparative Analysis Reveals Structural Control of Ubiquitylation Site Selection by BRCA1/BARD1

BRCA1/BARD1 is a chromatin-associated E3 ubiquitin ligase that ubiquitylates histone H2A to coordinate DNA damage repair, transcriptional repression, and genome stability. In Caenorhabditis elegans (C. elegans), the orthologous BRC-1/BRD-1 com-plex performs analogous functions but exhibits structural variation, most notably through an additional 11-residue loop in BRD-1 that is absent from human BARD1. Prior exper-iments indicate this worm-specific insertion promotes nucleosome engagement and may alter the preferred lysine target for ubiquitylation. Here, we provide a cross-species comparison by integrating computational and experimental investigation to clarify how a discrete structural variation can tune BRCA1-family ligase behavior and, consequently, chromatin regulation. In vitro ubiquitylation assays and mass spectrometry reveal BRC-1/BRD-1 ubiquitylate the C-terminal tail of histone H2A with less specificity than the human homologs. All-atom molecular dynamics simulations of both the C. elegans BRC-1/BRD-1-LET-70-Ubiquitin assembly and the human BRCA1/BARD1-UbcH5c-Ubiquitin complex in the presence of the nucleosome core particle uncover that the BRD-1 loop makes transient contacts with nucleosomal DNA and histone tails, thereby modulating the positioning and conformational flexibility of the bound E2 (ubiquitin-conjugating enzyme). Together, our results suggest that the BRD-1 loop alters the E3-E2 geometry, thereby altering ubiquitylation-site specificity.

biophysics↗

bioRxiv2026

Decoding the Conformational Dynamics and Hyperactivity of Histone H3K36 N-Methyltransferase in Oncogenic Mutations via tICA and Markov State Modeling

NSD2 is a histone methyltransferase that modifies lysine 36 in histone H3 (H3K36), playing a central role in chromatin organization and transcriptional regulation. Oncogenic mutations, such as E1099K and T1150A in NSD2, have been associated with hyperactive methylation, but the molecular mechanisms underlying this gain of function remain poorly understood. In this study, we performed all-atom molecular dynamics simulations on models of NSD2 bound to the nucleosome for the wild type (WT), E1099K, T1150A, and the E1099K/T1150A double mutant. Analysis of MD simulations reveals that the global dynamics of the enzymes remain unaltered upon mutations. The time-lagged independent component analysis (tICA) and Markov state modeling uncovered fundamental differences in free-energy landscapes among the variants. The WT NSD2 exhibited energetically and kinetically unfavorable transitions between the macrostates along with extended enzyme-substrate distances. On the other hand, the mutant systems demonstrate reduced SAM-H3K36 distances with modified energy landscapes that facilitate transitions or favor prolonged occupancy of catalytically competent states. Importantly, the mutations reorganize the network of intramolecular contacts around the catalytic site, SAM-binding pocket, and histone-binding interface, optimizing substance engagement geometry. These findings demonstrate that oncogenic mutations achieve hyperactivity through strategic reorganization of conformational dynamics rather than simple destabilization, balancing local flexibility with global stability to enhance catalytic efficiency. Our results provide mechanistic insights into NSD2 dysregulation in cancer and establish a framework to develop allosteric inhibitors that target the enzymes dynamic landscape.

biochemistry↗

bioRxiv2026

The structural mechanism of eukaryotic fluoride channel activation and inhibition by monovalent cations

Fluoride is an environmentally prevalent inhibitor of fungi, plants, and other eukaryotes. The fluoride exporter (FEX) is the major resistance mechanism that prevents intracellular fluoride accumulation in eukaryotes. FEX activity is sodium-dependent, but the mechanism for Na+ activation and the impact of other cations on FEX function remain poorly understood. Here, we show that sodium and lithium have different effects on channel activity. We leverage these differences to understand how monovalent cations regulate FEX. Functional assays in a reconstituted system show that lithium acts as a competitive antagonist of channel activation by competing with sodium for the central cation binding site. We further demonstrate that FEX inhibition by lithium has consequences in vivo, as lithium markedly reduces fluoride tolerance of yeast. A cryo-EM structure of Candida albicans FEX with Li+, together with molecular dynamics (MD) simulations, reveals the structural mechanism underlying cation-dependent FEX activity. Whereas sodium binding supports a dynamic structure with a broader pore radius, lithium coordination causes a more rigid conformation that is more compact in the channels vestibule. These changes perturb the tilt angle of a pore lining helix, and the alter the rotamer of a key phenylalanine in the pore, which together constrict the permeation pathway. In addition to providing general insight into the mechanism of fluoride channel regulation by monovalent cations, these results identify lithium as a previously unknown environmental antagonist of eukaryotic fluoride export and tie cellular fluoride stress tolerance to the abundance of additional ions in the cellular milieu.

biochemistry↗

bioRxiv2026

Asymmetry-induced distinct mechanisms and the transporting role of sodium in bacterial fluoride channel Fluc

Bacterial fluoride channels (Fluc) are exceptional in their ability to conduct fluoride ions at rapid rates while maintaining extraordinary selectivity, an unusual property among anion transporters. Fluc achieves this through an antiparallel homodimeric assembly that forms two asymmetric ion-conduction pores and hosts a centrally bound sodium ion of previously uncertain function. Despite extensive structural characterization, the molecular basis for Flucs dual-pore asymmetry, transport efficiency, and sodium involvement has remained unresolved. Here, we performed long-timescale molecular dynamics simulations under electrophysiological conditions to elucidate the mechanisms of fluoride translocation through each pore. Our results reveal two distinct conduction modes: Pore I operates via the Channsporter mechanism, characterized by single-ion transport; while Pore II follows the Multi-ion mechanism in which paired fluoride ions exploit electrostatic repulsion to achieve faster sequential translocations. These findings demonstrate that structural asymmetry gives rise to mechanistic specialization within the same homodimeric protein. We further propose that the central sodium ion serves as a dynamic cofactor in fluoride transport, coupling its vertical oscillation to fluoride movement in a pore-dependent manner. Together, our results establish a unified model of asymmetric dual-pore conduction in Fluc and introduce a broader paradigm in membrane transport biology, one where inherent structural asymmetry enables divergent yet coexisting mechanisms of ion conduction. SignificanceFluoride is a naturally occurring environmental ion that is toxic to bacteria at elevated concentrations, necessitating specialized export systems for survival. Bacteria encode two distinct fluoride exporters: the CLCF antiporter and the Fluc channel. The latter of which remains mechanistically unresolved due to its unusual dual-pore architecture and lack of close homologs. Using extensive molecular dynamics simulations, we dissect fluoride permeation through Fluc and show that its two antiparallel pores operate through distinct transport mechanisms. Besides, we uncover a previously unrecognized role for the central sodium ion as an essential cofactor that enables fluoride conduction. Together, these findings provide new mechanistic insight into fluoride export and clarify the functional asymmetry underlying Fluc-mediated transport.

biophysics↗

bioRxiv2025

The molecular basis of sodium-dependent fluoride export by the eukaryotic fluoride channel FEX

Much of life on Earth, including plants, fungi, and bacteria, evolved to resist toxic environmental fluoride. In eukaryotes, the major resistance mechanism is fluoride export by FEX proteins. Using electrophysiology and transport assays, we establish that FEX from plants and yeasts are fluoride channels whose activity depends on reversible sodium ion binding. A cryo-EM structure of FEX from Candida albicans, together with mutagenesis studies, reveals a fluoride permeation route through a single phenylalanine-lined pore. Molecular dynamics simulations demonstrate that a cation binding motif adjacent to the pore provides a stable sodium binding site that is accessible from the external aqueous solution. We propose that sodium gating resolves a major conundrum of channel-based fluoride efflux by preventing fluoride permeation under conditions of membrane depolarization. Comparison to bacterial fluoride channels (Flucs) provides a unique glimpse of the evolution of structural and mechanistic complexity in a membrane protein family with inverted repeat architecture.

biophysics↗

bioRxiv2025

Generative Landscapes and Dynamics to Design Multidomain Artificial Transmembrane Transporters

Protein design is challenging as it requires simultaneous consideration of interconnected factors, such as fold, dynamics, and function. These evolutionary constraints are encoded in protein sequences and can be learned through the latent generative landscape (LGL) framework to predict functional sequences by leveraging evolutionary patterns, enabling exploration of uncharted sequence space. By simulating designed proteins through molecular dynamics (MD), we gain deeper insights into the interdependencies governing structure and dynamics. We present a synergized workflow combining LGL with MD and biochemical characterization, allowing us to explore the sequence space effectively. This approach has been applied to design and characterize two artificial multidomain ATP-driven transmembrane copper transporters, with native-like functionality. This integrative approach proved effective in unraveling the intricate relationships between sequence, structure, and function.

biophysics↗

bioRxiv2024

Histone H3 Orchestrates the Ubiquitination of Nucleosomal H2A by BRCA1/BARD1-UbcH5c Complex

The Breast Cancer Associated Protein 1 (BRCA1) is a human tumor suppressor protein that commonly functions as ubiquitin ligase enzyme (E3) in the ubiquitination of the C-terminal H2A. BRCA1 enhances ubiquitin ligase activity by forming a heterodimeric complex with the BRCA1 Associated Ring Domain Protein (BARD1). The BRCA1/BARD1 complex works in concert with the ubiquitin-conjugating enzyme (UbcH5c or E2) to ubiquitinate one of the five lysines of the H2A C-terminal, ultimately promoting the repair of double-stranded DNA breaks. The mutations in the BRCA1-UbcH5c portion of the E3-E2 complex have been linked to breast and ovarian cancer. However, the mechanism of BRCA1/BARD1-UbcH5c complex ubiquitination at H2A is poorly understood, and the ubiquitination of exact lysine is debated. In this study, we sought to expand on the current research on H2A ubiquitination by using all-atom molecular dynamics simulations to model the BRCA1/BARD1-UbcH5c complex with the human ubiquitin protein (Ub). The Ub protein covalently bonds to the active site of E2, resulting in diminished flexibility of the E3-E2 complex with respect to the nucleosome core particle. The results of this study suggest a possible contribution of H3 in determining the preferred orientation of E2-Ub with respect to the H2A C-terminal lysines.

biochemistry↗

bioRxiv2023

Allosteric Modulation of YAP/TAZ-TEAD Interaction by Palmitoylation and Small Molecule Inhibitors

The Hippo signaling pathway is a highly conserved signaling network that plays a central role in regulating cellular growth, proliferation, and organ size. This pathway consists of a kinase cascade that integrates various upstream signals to control the activation or inactivation of YAP/TAZ proteins. Phosphorylated YAP/TAZ is sequestered in the cytoplasm; however, when the Hippo pathway is deactivated, they translocate into the nucleus, where they associate with TEAD transcription factors. This partnership is instrumental in regulating the transcription of pro-growth and anti-apoptotic genes. Thus, in many cancers, aberrantly hyperactivated YAP/TAZ promotes oncogenesis by contributing to cancer cell proliferation, metastasis, and therapy resistance. Because YAP and TAZ exert their oncogenic effects by binding with TEAD, it is critical to understand this key interaction to develop cancer therapeutics. Previous research has indicated that TEAD undergoes an auto-palmitoylation at a conserved cysteine, and small molecules that inhibit TEAD palmitoylation disrupt effective YAP/TAZ binding. However, how exactly palmitoylation contributes to YAP/TAZ-TEAD interactions and how the TEAD palmitoylation inhibitors disrupt this interaction remains unknown. Utilizing molecular dynamics simulations, our investigation not only provides a detailed atomistic insight into the YAP/TAZ-TEAD dynamics but also unveils that the inhibitor studied influences YAP and TAZ binding to TEAD in distinct manners. This discovery holds significant implications for the design and deployment of future molecular interventions targeting this interaction.

biophysics↗

bioRxiv2022

Uncovering the Mechanism of the Proton-Coupled Fluoride Transport in the CLCF Antiporter

Fluoride is a natural antibiotic abundantly present in the environment and, in micromolar concentrations, is able to inhibit enzymes necessary for bacteria to survive. However, as is the case with many antibiotics, bacteria have evolved resistance methods, including through the use of recently discovered membrane proteins. One such protein is the CLCF F-/H+ antiporter protein, a member of the CLC superfamily of anion-transport proteins, most notably known for their ability to transport chloride ions. While it possesses many similarities to the other CLC proteins, it also differs in several key ways, and though previous studies have examined this F- transporter, many questions are still left unanswered. To reveal details of the transport mechanism used by CLCF, we have employed molecular dynamics simulations and umbrella sampling calculations. Our results have led to several discoveries, including the mechanism of proton import and how it is able to aid in the fluoride export. Additionally, we have determined the role of the previously identified residues Glu118, Glu318, Met79, and Tyr396. This work is among the first computational studies of the CLCF F-/H+ antiporter and is the first to propose a mechanism which couples both the proton and anion transport.

biophysics↗

bioRxiv2022

Histone Tail Electrostatics Modulate E2-E3 Enzyme Dynamics: A Gateway to Regulate Ubiquitination Machinery

BRCA1 (BReast Cancer-Associated protein 1), a human tumor suppressor, plays a key role in genome stability and DNA repair. Heterodimerization of BRCA1 with BARD1 is important for its stability, maximal Ub ligase (E3) activity and cooperative activation of UbcH5c (E2). Recent studies demonstrate the importance of ubiquitination of the nucleosomal H2A C-terminal tail by BRCA1/BARD1-UbcH5c (E3-E2) in which its mutations inhibit ubiquitination, predispose cells to chromosomal instability and greatly increase the likelihood of breast and ovarian cancer development. Due to the lack of molecular-level insight on the flexible and disordered H2A C-tail, its ubiquitination mechanism by BRCA1/BARD1-UbcH5c and its function and relationship to cancer susceptibility remain elusive. Here, we use molecular dynamics simulations to provide molecular-level insights into the dynamics of the less-studied H2A C-tail and BRCA1/BARD1-UbcH5c on the nucleosome surface. Our results precisely identify the key interactions and residues that trigger conformational transitions of BRCA1/BARD1-UbcH5c, and characterize the important role of histone electrostatics in their dynamics. We show that the dynamics of the H2A C-tail, combined with the highly mobile UbcH5c, define the ubiquitination capacity. Furthermore, our data demonstrate a mechanistic basis for the probability of ubiquitination of C-tail lysines in the ordered and disordered regions. Altogether, the findings of this study will provide unrevealed insights into the mechanism of H2A C-tail ubiquitination and help us understand the communication between E2-E3 enzymes and nucleosome to regulate ubiquitination machinery, paving the way for the development of effective treatments for cancer and chronic pain. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=88 SRC="FIGDIR/small/505537v1_ufig1.gif" ALT="Figure 1"> View larger version (30K): org.highwire.dtl.DTLVardef@15c99e1org.highwire.dtl.DTLVardef@7ab5bdorg.highwire.dtl.DTLVardef@31b69forg.highwire.dtl.DTLVardef@1cca3b3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2017

Computational Investigation of O2 Diffusion Through an Intra-molecular Tunnel in AlkB; Influence of Polarization on O2 Transport

E. Coli AlkB catalyzes the direct dealkylation of various alkylated bases in damaged DNA. The diffusion of molecular Oxygen to the active site in AlkB is an essential step for the oxidative dealkylation activity. Despite detailed studies on the stepwise oxidation mechanism of AlkB, there is no conclusive picture of how O2 molecules reach the active site of the protein. Yu et al. (Nature, 439, 879) proposed the existence of an intra-molecular tunnel based on their initial crystal structures of AlkB. We have employed computational simulations to investigate possible migration pathways inside AlkB for O2 molecules. Extensive molecular dynamics (MD) simulations, including explicit ligand sampling and potential of mean force (PMF) calculations, have been performed to provide a microscopic description of the O2 delivery pathway in AlkB. Analysis of intra-molecular tunnels using the CAVER software indicates two possible pathways for O2 to diffuse into the AlkB active site. Explicit ligand sampling simulations suggests that only one of these tunnels provides a viable route. The free energy path for an oxygen molecule to travel along each of these tunnels has been determined with AMBER and AMOEBA. Both PMFs indicate passive transport of O2 from the surface of the protein. However, the inclusion of explicit polarization shows a very large barrier for diffusion of the co-substrate out of the active site, compared with the non-polarizable potential. In addition, our results suggest that the mutation of a conserved residue along the tunnel, Y178, has dramatic effects on the dynamics of AlkB and on the transport of O2 along the tunnel.

biochemistry↗