Biology subjects

Mani, N.

Publications and source records attributed to Mani, N..

9 recordsLinked to original sources

bioRxiv2026

Oligomannose Fc Glycans Reprogram the Energetic and Conformational Basis of CD16a Recognition

IgG1 Fc recognition by Fc{gamma}RIIIa/CD16a is a central determinant of antibody-dependent cellular cytotoxicity and is strongly regulated by Fc N297 glycosylation. While afucosylation and galactosylation have been extensively studied, the structural basis by which oligomannosidic Fc glycans modulate CD16a binding remains less clear, despite their prevalence in therapeutic antibodies and association with accelerated serum clearance. Here, we use all-atom molecular dynamics simulations to investigate how mannose-5 (M5) Fc glycosylation alters IgG1 Fc-CD16a recognition across Paired Biantennary (complex glycans on both Fc), asymmetric Unpaired (complex glycan on one Fc arm and M5 on the other), and Paired M5 glycoforms. Computed interaction energies reproduce the experimental trend that Paired M5 glycoforms bind CD16a less favorably than complex-type paired glycans, supporting the use of the simulations to interrogate the structural origin of this energetic hierarchy. Residue-wise energetic decomposition and contact analyses show that Paired M5 glycosylation redistributes energetic contributions away from the productive Fc-CD16a interface and reduces both protein-mediated and glycan-mediated physical contacts. Free energy surface analyses further reveal that Paired M5 systems sample broader, less stable receptor-bound conformational ensembles, while dynamic cross-correlation analysis shows reduced intra-domain and inter-domain coupling across the complex. Importantly, a single M5 glycan is sufficient to perturb productive recognition by increasing Fc-arm separation heterogeneity, reducing high-frequency protein contacts, and weakening long-range dynamic communication. Glycan identity on the receptor-proximal Fc arm emerges as a decisive determinant of binding, indicating that Fc glycan composition, pairing, and receptor-bound placement jointly encode CD16a recognition. Together, these findings provide a mechanistic framework for understanding how oligomannose Fc glycans remodel antibody-receptor engagement and suggest that asymmetric Fc glycosylation, combined with residue-level interface engineering, may offer new strategies for tuning therapeutic antibody effector function.

immunology↗

bioRxiv2026

Molecular Basis of Core Fucosylation-Dependent Modulation of IgG1-FcCD16a Binding

Core fucosylation of the IgG1 Fc N297 glycan is known to reduce binding affinity to the Fc{gamma}RIIIa (CD16a) receptor and attenuate antibody-dependent cellular cytotoxicity (ADCC), yet the structural mechanisms underlying this effect remain incompletely understood. Here, we use extensive all-atom molecular dynamics simulations to systematically investigate how Fc glycosylation modulates the structural, energetic, and dynamical landscape of the IgG1 Fc-CD16a complex across multiple systems with fucosylation and galactosylation. Relative binding free energy calculations reproduce experimentally established trends, showing that afucosylation consistently strengthens Fc-CD16a interactions. Mechanistically, dual fucosylation (on both Fc arms) increases inter-glycan packing between the Fc N297 glycans, restricts Fc glycan conformational sampling, and destabilizes the conformational organization of the CD16a N162 glycan. These glycan-mediated perturbations propagate to the protein interface. The result is reduced Fc-CD16a contact persistence, redistribution of energetically important residues away from the canonical binding interface, and broader, less stable receptor-bound conformational states. Dynamic cross-correlation analysis further reveals that afucosylated systems maintain substantially stronger coordinated motions across the Fc-CD16a assembly, whereas fucosylation disrupts long-range dynamic coupling between the receptor and antibody domains. Across these different energetic, structural, conformational, and dynamical readouts, fucosylation systematically shifts the Fc-CD16a assembly from a compact, interface-stabilized binding mode toward a more heterogeneous and weakly coupled receptor-bound ensemble. Together, our findings set forth a mechanistic basis for Fc glycosylation regulating receptor engagement through ensemble-level conformational and dynamical reorganization rather than simple local steric effects. These results provide mechanistic design principles for rational Fc glycoengineering and the development of therapeutic antibodies with enhanced effector functions. More broadly, this work highlights how glycan composition can be leveraged as a tunable molecular design parameter for engineering protein recognition, conformational stability, and immune effector function in therapeutic glycoproteins.

biophysics↗

bioRxiv2026

F.A.D.E. (Fully Agentic Drug Engine): A Conversational AI Platform for Drug Discovery

Drug discovery remains one of the costliest and most time-intensive endeavors in the pharmaceutical pipeline, with average development costs exceeding $2.3 billion per drug, timelines spanning more than a decade, and attrition rates above 90% in clinical trials. While computational methods have expanded the searchable chemical space, current pipelines remain fragmented and largely inaccessible to researchers without deep interdisciplinary expertise. Here we present F.A.D.E. (Fully Agentic Drug Engine), a multi-agent, open-source platform that converts natural language queries into potential drug candidates, substantially lowering the expertise barrier to advanced computational drug discovery. F.A.D.E. employs a three-branch hierarchical architecture that adapts to the level of available structural data for any protein target, integrating structure prediction, binding pocket detection, equivariant diffusion-based de novo ligand generation, and binding affinity estimation into a single automated pipeline. We validate F.A.D.E. on two structurally distinct targets: the epidermal growth factor receptor kinase domain (EGFR), a well-established oncology target, and cellular retinol-binding protein 1 (CRBP1), a lipid-binding protein involved in retinoid metabolism. For EGFR, our generated candidates achieved QED scores of 0.85 compared to 0.46 for the co-crystallised reference ligand, demonstrating marked improvement in predicted drug-likeness. Results across both targets confirm that F.A.D.E. can reliably generate chemically tractable, drug-like hit compounds across diverse protein classes from simple natural language input.

biophysics↗

bioRxiv2025

Natural and patient-derived mutations in BK polyomavirus VP1 reveal structural determinants of BC-loop dependent antibody escape

BK polyomavirus (BKPyV) reactivation poses a serious threat to both renal and hematopoietic transplant outcomes, with no approved antiviral therapies. Neutralizing monoclonal antibodies targeting the viral capsid protein VP1 have recently advanced into clinical development, driven by promising in vitro potency. Since naturally elicited neutralizing antibodies arise following infection, we examined whether host immune pressure may have shaped the VP1 surface, with implications for therapeutic application. To probe the impact of VP1 coevolution on therapeutic engagement, we evaluated two neutralizing antibody formats: the clinical-stage monoclonal antibody 319C07 and nanobodies VHH16 and VHH17. Our X-ray crystallographic analysis of the VP1 pentameric core in complex with antibody fragments showed a convergent receptor- mimetic engagement of the sialic acid-binding cleft, mapping to a shared epitope centered around the VP1 BC-loop. Analysis of over 900 BKPyV VP1 sequences from public databases, combined with longitudinal VP1 sequence data from transplant recipients, highlighted widespread pre-existing diversity at the BC-loop contact sites, underscoring the evolutionary adaptability of BKPyV at this antigenic surface that may compromise therapeutic recognition. Through integrated mutagenesis and binding analyses, we show that circulating BC-loop mutations, including single amino acid substitutions, are sufficient to abrogate 319C07 binding and neutralization. Nanobodies VHH16 and VHH17, selected for their compact size and potential to access intrarenal sites of BKPyV replication, also exhibited complete loss of binding across multiple clinically observed VP1 variants. Our findings may offer a mechanistic framework to interpret the translational gap between in vitro neutralization and clinical efficacy of these biologics. As part of ongoing efforts to target VP1, we demonstrate, for the first time, small molecules with nanomolar to sub-nanomolar affinities against BKPyV VP1 variants including potent binding to JCPyV VP1. Our work paves a path towards a new class of antiviral strategies that could block replication at intracellular replication sites, provide a higher genetic barrier to resistance and the potential to address both viral reactivation and persistent, high- burden viremia in transplant settings.

microbiology↗

bioRxiv2025

Cleaved vs. Uncleaved: How Furin Cleavage Reshapes the Conformational Landscape of SARS-CoV-2 Spike

The SARS-CoV-2 Spike protein is the primary target for vaccine design, with immunogens typically engineered to enhance stability by introducing proline mutations (2P) and mutating or deleting the Furin Cleavage Site (FCS). While these modifications improve structural integrity, studies suggest that furin cleavage can play a functional role in Spike protein dynamics, potentially enhancing ACE2 receptor binding. However, the impact of this cleavage on the unbound form of the Spike protein remains unclear. In this study, we use extensive all-atom molecular dynamics (MD) simulations to compare the structural and dynamic properties of cleaved and uncleaved Spike proteins in their pre-fusion, unbound state. Our results show that Furin cleavage significantly alters allosteric communication within the protein, increasing correlated motions between the Receptor Binding Domain (RBD) and N-terminal Domain (NTD), which may facilitate receptor engagement. Principal Component Analysis (PCA) reveals that the cleaved and uncleaved Spike proteins sample distinct conformational landscapes, with the cleaved form displaying enhanced flexibility and a broader range of RBD tilt angles. Additionally, Furin cleavage primes the S2 subunit by expanding the central helix, potentially influencing the transition to the post-fusion state. Glycan clustering patterns further suggest an adaptive structural response to cleavage, particularly in the NTD and RBD regions. These findings highlight the potential functional consequences of FCS deletion in immunogen design and underscore the importance of considering the native cleavage state in vaccine and therapeutic development.

biophysics↗

bioRxiv2022

Spatially controlled microtubule nucleation and organization from crosslinker MAP65 condensates

Microtubule organization in cells is essential for the internal structure and coordination of events of intracellular transport, mitosis, and cell motility. For many cell types, microtubule organization is dominated by centrosomal nucleation that use gamma-tubulin to template filaments. Yet, some cell types lack centrosomes or centrioles, such as plant cells. Instead, microtubules nucleate from regions with high concentrations of microtubule binding and nucleating proteins. A mechanism that can drive high local concentrations of nucleators is liquid-liquid phase separation of proteins with intrinsically disordered regions. Here, we report that the plant microtubule nucleator and crosslinking protein, MAP65-1, can form phase separated condensates at physiological salt and temperature without extra crowding agents. These condensates are liquid at first and can mature to gel-like phases over time and with different environmental conditions. We show that these condensates can nucleate and grow microtubule bundles that form asters, regardless of the viscoelasticity of the condensate. When gel-like droplets nucleate and grow asters from a shell of tubulin at the surface, the microtubules are able to re-fluidize the MAP65 condensate. Condensate-induced cytoskeletal formation could be a universal mechanism for organization of the microtubule and actin cytoskeletons in all cell types, especially cells without centrosomes.

biophysics↗

bioRxiv2021

Cytoskeletal regulation of a transcription factor by DNA mimicry

A long-established strategy for transcription regulation is the tethering of transcription factors to cellular membranes. In contrast, the principal effectors of Hedgehog signaling, the Gli transcription factors, are regulated by microtubules in the primary cilium and the cytoplasm. How Gli is tethered to microtubules remains unclear. We uncover DNA mimicry by the ciliary kinesin Kif7 as a mechanism for the recruitment of Gli to microtubules, revealing a new mode of tethering a DNA-binding protein to the cytoskeleton. Gli increases the Kif7-microtubule affinity and consequently modulates the localization of both proteins to microtubules and the cilium tip. Thus, the kinesin-microtubule system is not a passive Gli tether but a regulatable platform tuned by the kinesin-transcription factor interaction. We re-tooled the unique DNA-mimicry-based Gli-Kif7 interaction for inhibiting the nuclear and cilium localization of Gli. This strategy can be potentially exploited for downregulating erroneously activated Gli in human cancers.

cell biology↗

bioRxiv2020

Confinement Discerns Swarmers from Planktonic Bacteria

Powered by flagella, many bacterial species exhibit collective motion on a solid surface commonly known as swarming. As a natural example of active matter, swarming is also an essential biological phenotype associated with virulence, chemotaxis, and host pathogenesis. Physical changes like cell elongation and hyper flagellation have been shown to accompany the swarming phenotype. However, less noticeable, are the contrasts of collective motion between the swarming cells and the planktonic cells of comparable cell density. Here, we show that confining bacterial movement in designed dimensions allows distinguishing bacterial swarming from collective swimming. We found that on a soft agar plate, a novel bacterial strain Enterobacter sp. SM3 exhibited different motion patterns in swarming and planktonic states when confined to circular microwells of a specific range of sizes. When the confinement diameter was between 40 m and 90 m, swarming SM3 formed a single swirl motion pattern in the microwells whereas planktonic SM3 showed multiple swirls. Similar differential behavior is observed across a range of randomly selected gram-negative bacteria. We hypothesize that the "rafting behavior" of the swarming bacteria upon dilution might account for the motion pattern difference. We verified our conjectures via numerical simulations where swarming cells are modeled with lower repulsion and more substantial alignment force. The novel technical approach enabled us to observe swarming on a non-agar tissue surface for the first time. Our work provides the basis for characterizing bacterial swarming under more sophisticated environments, such as polymicrobial swarmer detection, and in vivo swarming exploration.

biophysics↗

bioRxiv2020

Differential Regulation of Single Microtubules and Cross-linked Bundles by a Minimal Three-Protein Module

A remarkable feature of the microtubule cytoskeleton is co-existence of sub-populations having different dynamic properties. A prominent example is the anaphase spindle, where stable antiparallel bundles exist alongside dynamic microtubules and provide spatial cues for cytokinesis. How are dynamics of spatially proximal arrays differentially regulated? We reconstitute a minimal system of three midzone proteins: microtubule-crosslinker PRC1, and its interactors CLASP1 and Kif4A, proteins that promote and suppress microtubule elongation, respectively. We find their collective activity promotes elongation of single microtubules, while simultaneously stalling polymerization of crosslinked bundles. This differentiation arises from (i) Strong rescue activity of CLASP1, which overcomes weaker effects of Kif4A on single microtubules, (ii) Lower microtubule and PRC1-binding affinity of CLASP1, which permit dominance of Kif4A at overlaps. In addition to canonical mechanisms where antagonistic regulators set microtubule lengths, our findings illuminate design principles by which collective regulator activity creates microenvironments of arrays with distinct dynamic properties.

cell biology↗