Biology subjects

Barbar, E. J.

Publications and source records attributed to Barbar, E. J..

8 recordsLinked to original sources

bioRxiv2025

Weak Motifs, Strong Complex: KANK1 Uses Cooperative Multivalency with the Hub-Protein LC8 to Bridge Cytoskeletal Complexes

The cortically anchored adaptor KANK1 organizes microtubules at focal adhesions through a long, intrinsically disordered linker (L2), yet how this linker spans the [~]35-50 nm membrane-microtubule gap is unclear. Here, we combine in-cell, biochemical, and biophysical assays, predictions of motif interaction and multivalent assembly using AlphaFold, and structural analysis by electron microscopy to show that the hub protein LC8, which binds more than 100 clients, converts the intrinsically disordered 600 amino acid L2 into an elongated, multivalent, rod-like assembly. In contrast, isolated motif peptides fail to bind LC8 at physiologically relevant concentrations, indicating that strong complex formation arises from cooperativity among multiple weak sites. These results establish LC8 as a molecular switch that rigidifies and extends KANK1 L2 via distributed weak motifs and short linkers. This interaction produces compositionally homogeneous yet conformationally adaptable rods, long enough to bridge the membrane-microtubule gap, resolving the paradox. This work expands the LC8 binding repertoire, reveals design principles for multivalent assembly, and suggests a generalizable strategy for tuning length, rigidity, and flexibility in large protein architectures.

biophysics↗

bioRxiv2025

Negative Cooperativity in a Polybivalent Complex Allows the Prevalence of a Partial Bound State.

The central region of the cytoplasmic dynein complex, comprising the intermediate chain (IC) and two light chains (LC8 and Tctex1), has eluded thorough quantitative characterization due to its participation in a highly coupled seven-state binding network. Although isothermal titration calorimetry (ITC) is the gold standard for measuring binding thermodynamics, conventional analyses are limited to simple interaction schemes because individual isotherms contain insufficient information to resolve complex reaction networks. Here, we overcome this limitation by combining extensive experimental sampling with hierarchical Bayesian inference. We collected 39 ITC isotherms spanning eight experiment types and developed a global Bayesian framework integrating multiple datasets while explicitly accounting for concentration uncertainty. Using this approach, we fit the complete dataset to a mechanistic seven-state model, estimating 190 parameters, including 12 thermodynamic parameters while marginalizing over 178 nuisance parameters. Remarkably, this strategy yields 95% confidence intervals for thermodynamic values as narrow as 0.05 kcal/mol and back-propagates to nanomolar precision in effective concentrations, even when experimental concentrations are in the hundreds of micromolar. The resulting thermodynamic landscape enables predictive modelling of assembly populations under different scenarios, including binding states inaccessible to standard ITC analyses. These results reveal previously unrecognized binding states that may play key roles in dynein cargo attachment and release. More broadly, this work reveals a form of "multi-cooperativity" governing dynein assembly and demonstrates how intensive experimentation coupled with modern statistical tools can resolve complex molecular systems beyond the reach of traditional biophysical techniques. Significance StatementLarge, complex mechanistic processes have remained difficult to fully characterize, which limits interpretability of the underlying biology. We utilize a large dataset of 39 complementary experiments to fully characterize a seven-state system using Bayesian inference. This process achieves impressively precise fits with 0.05 kcal/mol width confidence intervals. The high precision enables assessment of simultaneous positive and negative cooperativity in the assembly of the dynein intermediate chain with its light-chain partners. Simulation of state populations suggests that this balancing cooperativity is finely tuned to allow access to a half-bound state which has been previously inaccessible quantitatively. Our approach is broadly applicable and supports an emerging principle of molecular regulation--negative cooperativity as a strategy for tuning responsiveness and dynamic control.

biophysics↗

bioRxiv2025

Improving parameter inference by resolving Bayesian prior ambiguity via multi-dataset analysis: Application to isothermal titration calorimetry

Isothermal titration calorimetry (ITC) is a powerful technique for probing biomolecular interactions. However, accurate determination of binding parameters--such as enthalpy and free energy--as well as associated uncertainties can be hindered by noise and concentration variability. Notably, the mathematical ambiguity surrounding analyte concentrations in standard binding models intrinsically limits the precision with which binding parameters, particularly binding enthalpies, can be determined. Here, we present a Bayesian pipeline that resolves this ambiguity by combining two key strategies: simultaneous analysis of multiple ITC datasets and a hierarchical Bayesian treatment of analyte concentration priors. This dual approach not only lifts the degeneracy inherent in single-dataset studies but also removes an ambiguity typically present in Bayesian analysis by self-consistently refining concentration estimates, ensuring optimal joint inference of binding parameters and concentrations. Using modern Monte Carlo techniques enables our pipeline to provide robust posterior sampling for more than 10 datasets and 40 total parameters. We validate the approach with synthetic ITC datasets for single- and multi-site binding models and apply it to experimental data, including 14 datasets for 1:1 binding of Mg(II) to the chelator EDTA and multiple datasets of the hub protein LC8 with diverse binding partners. This work serves as a foundation for improving the precision of binding constants using multiple ITC datasets, while providing a systematic framework for assessing the reliability of experimental concentration estimates, paving the way for more accurate biomolecular interaction studies.

biophysics↗

bioRxiv2024

LC8 enhances 53BP1 foci through heterogeneous bridging of 53BP1 oligomers

53BP1 is a key player in DNA repair and together with BRCA1 regulate selection of DNA double strand break repair mechanisms. Localization of DNA repair factors to sites of DNA damage by 53BP1 is controlled by its oligomerization domain (OD) and binding to LC8, a hub protein that functions to dimerize >100 clients. Here we show that 53BP1 OD is a trimer, an unusual finding for LC8 clients which are all dimers or tetramers. As a trimer, 53BP1 forms a heterogeneous mixture of complexes when bound to dimeric LC8 with the largest mass corresponding to a dimer-of-trimers bridged by 3 LC8 dimers. Analytical ultracentrifugation and isothermal titration calorimetry demonstrate that only the second of the three LC8 recognition motifs is necessary for a stable bridged complex. The stability of the bridged complex is tuned by multivalency, binding specificity of the second LC8 site, and the length of the linker separating the LC8 binding domain and OD. 53BP1 mutants deficient in bridged species fail to impact 53BP1 focus formation in human cell culture studies, suggesting that the primary role of LC8 is to bridge 53BP1 trimers which in turn promotes recruitment of 53BP1 at sites of DNA damage. We propose that the formation of higher-order oligomers of 53BP1 explains how LC8 elicits an improvement in 53BP1 foci and affects the structure and functions of 53BP1.

biophysics↗

bioRxiv2023

RNA structure and multiple weak interactions balance the interplay between RNA binding and phase separation of SARS-CoV-2 nucleocapsid

The nucleocapsid (N) protein of SARS-CoV-2 binds viral RNA, condensing it inside the virion, and phase separating with RNA to form liquid-liquid condensates. There is little consensus on what differentiates sequence-independent N-RNA interactions in the virion or in liquid droplets from those with specific genomic RNA motifs necessary for viral function inside infected cells. To identify the RNA structures and the N domains responsible for specific interactions and phase separation, we use the first 1000nt of viral RNA and short RNA segments designed as models for single-stranded and paired RNA. Binding affinities estimated from fluorescence anisotropy of these RNAs to the two folded domains of N (the NTD and CTD) and comparison to full-length N demonstrate that the NTD binds preferentially to single-stranded RNA, and while it is the primary RNA binding site, it is not essential to phase separation. Nuclear magnetic resonance spectroscopy identifies two RNA binding sites on the NTD: a previously characterized site and an additional although weaker RNA-binding face that becomes prominent when binding to the primary site is weak, such as with dsRNA or a binding-impaired mutant. Phase separation assays of nucleocapsid domains with different RNA structures support a model where multiple weak interactions, such as with the CTD or the NTDs secondary face promote phase separation, while strong, specific interactions do not. These studies indicate that both strong and multivalent weak N-RNA interactions underlie the multifunctional abilities of N. SignificanceThe nucleocapsid protein of the SARS-CoV-2 coronavirus binds to viral RNA, both to protect and condense it inside the viral particle and to facilitate viral transcription inside infected host cells. Evidence suggests that variations in RNA structure impact how and where it binds to the nucleocapsid, but these differences are not well understood at a structural level. Using nuclear magnetic resonance spectroscopy, we examine the interactions between each folded domain of the nucleocapsid and different RNA structures. Binding affinities and NMR chemical shift profiles demonstrate that binding between the N-terminal domain and single stranded RNA is driven by strong interactions at a specific site, while multiple weak nonspecific interactions at newly discovered sites lead to phase separation and RNA condensation.

biophysics↗

bioRxiv2022

Multivalency, autoinhibition, and protein disorder in the regulation of interactions of dynein intermediate chain with dynactin and the nuclear distribution protein

Cytoplasmic dynein plays crucial roles in the intracellular transport of organelles and other cargoes. Central to dynein function is the intrinsically disordered N-terminal domain of dynein intermediate chain (IC), which binds the three dimeric dynein light chains at multivalent sites, and dynactin p150Glued and nuclear distribution protein (NudE) at overlapping sites. The disorder in IC has hindered cryo-electron microscopy and X-ray crystallography studies of its structure and interactions. Here we use a suite of biophysical methods to reveal how multivalent binding of the three light chains regulate IC interactions with p150Glued and NudE. Using the N-terminal domain or the full-length IC from Chaetomium thermophilum, a tractable species to interrogate IC interactions, we identify a significant reduction in ICs binding affinity for p150Glued and a loss of binding to NudE in contrast to the tight binding observed with small IC constructs. We attribute this difference to autoinhibition caused by strong long-range intramolecular interactions that cover ICs N-terminal single -helix, the site for p150Glued and NudE binding. Reconstitution of IC subcomplexes demonstrate that autoinhibition is differentially regulated by light chains binding underscoring their importance both in assembly and organization of IC, and in selection between multiple binding partners at the same site.

biophysics↗

bioRxiv2021

Dimerisation of the PICTS complex via LC8/Cut-up drives co-transcriptional transposon silencing in Drosophila

In animal gonads, the PIWI-interacting RNA (piRNA) pathway guards genome integrity in part through the co-transcriptional gene silencing of transposon insertions. In Drosophila ovaries, piRNA-loaded Piwi detects nascent transposon transcripts and instructs heterochromatin formation through the Panoramix-induced co-transcriptional silencing (PICTS) complex, containing Panoramix, Nxf2 and Nxt1. Here, we report that the highly conserved dynein light chain LC8/Cut-up (Ctp) is an essential component of the PICTS complex. Loss of Ctp results in transposon de-repression and a reduction in repressive chromatin marks specifically at transposon loci. In turn, Ctp can enforce transcriptional silencing when artificially recruited to RNA and DNA reporters. We show that Ctp drives dimerisation of the PICTS complex through its interaction with conserved motifs within Panoramix. Artificial dimerisation of Panoramix bypasses the necessity for its interaction with Ctp, demonstrating that conscription of a protein from a ubiquitous cellular machinery has fulfilled a fundamental requirement for a transposon silencing complex.

molecular biology↗

bioRxiv2019

Cataract-associated deamidations on the surface of γS-crystallin increase protein unfolding and flexibility at distant regions

Deamidation is a major age-related modification in the human lens that is highly prevalent in crystallins isolated from cataractous lenses. However, the mechanism by which deamidation causes proteins to become insoluble is not known, because of only subtle structural changes observed in vitro. We have identified Asn14 and Asn76 of {gamma}S-crystallin as highly deamidated in insoluble proteins. These sites are on the surface of the N-terminal domain and were mimicked by replacing the Asn with Asp residues. We used heteronuclear NMR spectroscopy to measure their amide hydrogen exchange and 15N relaxation dynamics to identify regions with significantly increased dynamics compared to wildtype-{gamma}S. Changes in dynamics were localized to the C-terminal domain, particularly to helix and surface loops distant from the mutation sites. Thus, a potential mechanism for {gamma}S deamidation-induced insolubilization in cataractous lenses is altered dynamics due to local regions of unfolding and increased flexibility.

biophysics↗