Biology subjects

Tavakoli, A.

Publications and source records attributed to Tavakoli, A..

8 recordsLinked to original sources

bioRxiv2025

Sequence-Based Generative AI-Guided Design of Versatile Tryptophan Synthases

Enzymes offer unparalleled selectivity and sustainability for chemical synthesis, yet their widespread industrial application is often hindered by the slow and uncertain process of discovering and optimizing suitable biocatalysts. While directed evolution remains the gold standard for enzyme optimization, its success hinges on the availability of a starting enzyme with measurable activity, a persistent bottleneck for many desired functions. Designing libraries likely to contain such functional starting points remains a major challenge. In this work, we use the GenSLM protein language model (PLM) along with a series of filters to generate novel sequences of the {beta}-subunit of tryptophan synthase (TrpB) that express in Escherichia coli, are stable, and are catalytically active in the absence of a TrpA partner. Many generated TrpBs also demonstrated significant substrate promiscuity, accepting non-canonical substrates typically inaccessible to natural TrpBs. Remarkably, several outperformed both natural and laboratory-optimized TrpBs on native and non-canonical substrates. Comparative analysis of the most active and promiscuous generated TrpB and its closest natural homolog confirmed that this enhanced functional versatility does not stem from the natural enzyme, highlighting the creative potential of generative models. Our results demonstrate that the model can generate enzymes which not only preserve natural structure and function but also acquire non-natural properties, establishing PLMs as powerful tools for biocatalyst discovery and engineering, with the potential in some cases to bypass further optimization.

biochemistry↗

bioRxiv2025

The cryo-EM structure of mouse radial spoke 3 reveals a unique metabolic and regulatory hub in cilia

Cilia are complex, microtubule-based organelles that protrude from many eukaryotic cells and have important roles in sensing, signaling, and motility. Recent studies have revealed the atomic structures of many multi-component ciliary complexes, providing new insights into their mechanisms of action that are vital for cilias biological functions. However, little is known about the structure, proteome, and function of full-length radial spoke 3 (RS3), which is distinct from the structurally well-characterized RS1 and RS2. Radial spokes are conserved megadalton complexes that transmit mechanochemical signals from the central pair of microtubules to the dynein motors, thereby coordinating ciliary motility. Here, we combined cryo-electron microscopic single-particle reconstruction, cryo-electron tomography (cryo-ET), proteomic analysis, and computational modeling to determine the 3D structure and atomic model of RS3 from mouse respiratory cilia. Our structure reveals all protein components of RS3, including regulatory and metabolic enzymes, such as a protein kinase A subunit, adenylate kinases and malate dehydrogenases. We have confirmed the important role of adenylate kinase 7 in RS3 by cryo-ET analyses of respiratory cilia in AK7-deficient mice, which display primary ciliary dyskinesia. Our findings suggest that RS3 is an important regulatory hub and cluster of metabolic proteins that helps to maintain ATP at the levels required for sustained dynein motor activity and ciliary beating. This work advances our understanding of the structure and function of RS3 in ciliary motility and provides insights into the etiology of ciliopathies.

biophysics↗

bioRxiv2025

Kinocilia of Vestibular Hair Cells: Bridging Structural and Functional Traits of Primary and Motile Cilia

Vestibular hair cells (HCs) convert gravitational and head motion cues into neural signals through mechanotransduction, mediated by the hair bundle--a mechanically integrated organelle composed of stereocilia and a kinocilium. The kinocilium, a specialized form of primary cilium, remains incompletely defined in structure, molecular composition, and function. To elucidate its characteristics, we conducted single-cell RNA sequencing of adult vestibular and cochlear HCs, uncovering a selective enrichment of primary and motile cilia-associated genes in vestibular HCs, particularly those related to the axonemal repeat complex. This enrichment of orthologous axonemal-related genes was conserved in zebrafish and human vestibular HCs, indicating a shared molecular architecture. Immunostaining validated the expression of key motile cilia markers in vestibular kinocilia. Moreover, live imaging of bullfrog and mouse HCs from crista ampullaris revealed spontaneous kinociliary motion. Together, these findings define the kinocilium as a unique organelle with molecular features of primary and motile cilia and suggest its previously unknown role as an active, force-generating element within the hair bundle.

neuroscience↗

bioRxiv2024

Hemifusomes and Interacting Proteolipid Nanodroplets: Formation of a Novel Cellular Organelle Complex

Within cells, vesicle fusion, scission, and the formation of intraluminal vesicles are critical processes that facilitate traffic, degradation, and recycling of cellular components, and maintenance of cellular homeostasis. Despite significant advancements in elucidating the molecular mechanisms that drive these dynamic processes, the direct in situ visualization of membrane remodeling intermediates remains challenging. Here, through the application of cryo-electron tomography in mammalian cells, we have identified a previously undescribed vesicular organelle complex with unique membrane topology: heterotypic hemifused vesicles that share extended hemifusion diaphragms (HDs) with a 42 nm proteolipid nanodroplet (PND) at their rim. We have termed these organelle complexes "hemifusomes". The HDs of hemifusomes exhibit a range of sizes and curvatures, including the formation of lens-shaped compartments encapsulated within the membrane bilayer. The morphological diversity of the lens-shaped vesicle aligns with a step-wise process of their intraluminal budding, ultimately leading to their scission and the generation of intraluminal vesicles. We propose that hemifusomes function as versatile platforms for protein and lipid sorting and as central hubs for the biogenesis of intraluminal vesicles and the formation of multivesicular bodies.

cell biology↗

bioRxiv2023

nELISA: A high-throughput, high-plex platform enables quantitative profiling of the secretome

We present the nELISA, a high-throughput, high-fidelity, and high-plex protein profiling platform. DNA oligonucleotides are used to pre-assemble antibody pairs on spectrally encoded microparticles and perform displacement-mediated detection. Spatial separation between non-cognate antibodies prevents the rise of reagent-driven cross-reactivity, while read-out is performed cost-efficiently and at high-throughput using flow cytometry. nELISA can measure both protein concentration and their post-translational modifications. We assembled an inflammatory panel of 191 targets that were multiplexed without cross-reactivity nor impact on performance vs 1-plex signals, with sensitivities as low as 0.1 pg/mL and measurements spanning 7 orders of magnitude. We then performed a large-scale inflammatory-secretome perturbation screen of peripheral blood mononuclear cells (PBMCs), with cytokines as both perturbagens and read-outs, measuring 7,392 samples and generating [~]1.4M protein data points in under a week; a significant advance in throughput compared to other highly multiplexed immunoassays. We uncovered 447 significant cytokine responses, including multiple putatively novel ones, that were conserved across donors and stimulation conditions. We validate nELISA for phenotypic screening, where its capacity to faithfully report hundreds of proteins make it a powerful tool across multiple stages of drug discovery.

bioengineering↗

bioRxiv2020

Tethering-facilitated DNA 'opening' and complementary roles of β-hairpin motifs in the Rad4/XPC DNA damage sensor protein

XPC/Rad4 initiates eukaryotic nucleotide excision repair on structurally diverse helix-destabilizing/distorting DNA lesions by selectively opening these sites while rapidly diffusing along undamaged DNA. Previous structural studies showed that Rad4, when tethered to DNA, could also open undamaged DNA, suggesting a kinetic gating mechanism whereby lesion discrimination relied on efficient opening versus diffusion. However, solution studies in support of such a mechanism were lacking and how opening is brought about remained unclear. Here, we present crystal structures and fluorescence-based conformational analyses on tethered complexes, showing that Rad4 can indeed open undamaged DNA in solution and that such opening can largely occur without one or the other of the {beta}-hairpin motifs in the BHD2 or BHD3 domains. Notably, the Rad4-bound open DNA adopts multiple conformations in solution notwithstanding the DNAs original structure or the {beta}-hairpins. Molecular dynamics simulations reveal compensatory roles of the {beta}-hairpins, which may render robustness in dealing with and opening diverse lesions. Our study showcases how fluorescence-based studies can be used to obtain information complementary to ensemble structural studies. The tethering-facilitated DNA opening of undamaged sites and the dynamic nature of open DNA may shed light on how the protein functions within and beyond NER in cells.

biochemistry↗

bioRxiv2020

Light-induced modulation of DNA recognition by the Rad4/XPC damage sensor protein

Biomolecular structural changes upon binding/unbinding are key to their functions. However, characterization of such dynamical processes with high spatial and temporal resolutions is difficult as it requires ways to rapidly trigger the assembly/disassembly as well as ways to monitor the structural changes over time. Recently, various chemical strategies have been developed to use light to trigger changes in oligonucleotide structures, thereby their activities. Here we report that photoswitchable DNA can be used to modulate the DNA binding of the Rad4/XPC DNA repair complex using light. Rad4/XPC specifically binds to diverse helix-destabilizing/distorting lesions including bulky organic adducts and functions as a key initiator for the eukaryotic nucleotide excision repair (NER) pathway. We show that the 6-nitropiperonyloxymethyl (NPOM)-modified DNA is recognized by the Rad4 protein as a specific substrate and that the specific binding can be abolished by light-induced cleavage of the NPOM group from DNA in a dose-dependent manner. Fluorescence lifetime-based analyses of the DNA conformations suggest that free NPOM-DNA retains B-DNA-like conformations despite its bulky NPOM adduct, but Rad4-binding renders it to be heterogeneously distorted. Subsequent extensive conformational searches and molecular dynamics simulations demonstrate that NPOM in DNA can be housed in the major groove of the DNA, with stacking interactions among the nucleotide pairs remaining largely unperturbed and thus retaining overall B-DNA conformation. Our work suggests that photoactivable DNA can be used as a DNA lesion surrogate to study DNA repair mechanisms such as nucleotide excision repair. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=103 SRC="FIGDIR/small/313114v1_ufig1.gif" ALT="Figure 1"> View larger version (34K): org.highwire.dtl.DTLVardef@1f31fc7org.highwire.dtl.DTLVardef@ab512dorg.highwire.dtl.DTLVardef@15e8493org.highwire.dtl.DTLVardef@51f58d_HPS_FORMAT_FIGEXP M_FIG Graphical Summary This work shows that a photolabile 6-nitropiperonyloxymethyl (NPOM)-modified DNA is specifically recognized by the Rad4/XPC damage sensor protein complex that initiates the nucleotide excision repair pathway; light-induced cleavage of NPOM abolishes the specific binding to Rad4/XPC. C_FIG

biochemistry↗

bioRxiv2020

Impact of DNA sequences in the DNA duplex opening by the Rad4/XPC nucleotide excision repair complex

Rad4/XPC is a key DNA damage sensor for nucleotide excision repair (NER) in eukaryotes. Rad4/XPC recognizes diverse bulky lesions by flipping out two lesion-containing nucleotide pairs and inserting a {beta}-hairpin from the BHD3 domain ({beta}-hairpin3) into the DNA duplex. We have previously observed that Rad4 can form the same open structure when covalently tethered to a normal DNA sequence containing consecutive C/Gs (CCC/GGG) and that a similar open-like structure can be formed even when the {beta}-hairpin3 is lacking. Here, we report a crystal structure of the {Delta}{beta}-hairpin3 mutant tethered to a sequence containing alternating C/Gs (CGC/GCG). In contrast to the previous structures, Rad4 bound to CGC/GCG in a 180{degrees}-reversed manner, capping the end of the duplex without flipping out the nucleotides. MD simulations showed that CGC/GCG was inherently less openable than CCC/GGG and that Rad4 failed to engage with its minor groove, a hallmark of productive binding towards opening. These results reveal that DNA sequences significantly influence the thermodynamic barrier for DNA opening by Rad4, which may render certain DNA structures/sequences resistant to opening despite a long residence time of Rad4. The reverse- mode may indicate unproductive binding for NER whereas the DNA end-binding may hint at Rad4/XPCs functions beyond NER.

biochemistry↗