Biology subjects

Al-Bassam, J.

Publications and source records attributed to Al-Bassam, J..

10 recordsLinked to original sources

bioRxiv2025

The Structural Basis of alpha/beta-tubulin Assembly and Disassembly by Tubulin Cofactors

Microtubules polymerize from cytoplasmic pools of soluble {beta}-tubulin heterodimers that support diverse cellular functions. The tubulin cofactors, TBCC, TBCD, TBCE, and the Arl2 GTPase, form TBC-DEG assemblies that regulate {beta}-tubulin assembly and disassembly from - and {beta}-tubulins, yet their underlying mechanisms remain incompletely understood. Here, we reconstitute the human TBC-DE and TBC-DEG assemblies from eukaryotic cells co-purified with monomeric {beta}-tubulin intermediates and determine their cryo-EM structures. The structures reveal that TBC-DEG disassembles {beta}-tubulin by releasing -tubulin through a lever-arm-like rotation in TBCE coupled to major conformational change in Arl2 upon its nucleotide release, while TBCD tightly holds {beta}-tubulin. TBCD dissociates -tubulin by refolding the {beta}-tubulin H10-S8 loop at its intradimer interface. The TBC-DEG-{beta}-tubulin or TBC-DE-{beta}-tubulin assemblies undergo extensive back-to-back dimerization mediated by {beta}-{beta}-tubulin homodimers, formed through their dissociated H8 helices at unoccupied intradimer interfaces. Structural comparisons demonstrate that the TBCE mechanical rotation, driven by the Arl2 GTPase cycle, either delivers -tubulin or removes it from beneath the TBCD-bound {beta}-tubulin and is directionally regulated by TBCC stabilizing {beta}-tubulin interfaces. Our findings suggest that TBC-DEG/TBCC catalyzing heterodimerization of -tubulin with {beta}-tubulin may have evolved to counteract the {beta}-tubulin intrinsic tendency to form off-pathway toxic homodimers through its exposed -tubulin-binding intradimer interface.

biochemistry↗

bioRxiv2025

Structural Basis of Kinesin-1 Autoinhibition and Its Control of Microtubule-Based Motility

Kinesin-1 was the first identified microtubule-based motor protein that drives anterograde intracellular transport of diverse cargoes in eukaryotic cells. Improper regulation and kinesin-1 defects are implicated in multiple neurological disorders as well as pathogens hijacks kinesin-1 to deliver their cargoes. Despite its importance, the molecular mechanisms governing kinesin-1 regulation and activation remains poorly understood. Here, we report the cryo-EM structure of the autoinhibited kinesin-1 heterotetramer and validate it using crosslinking mass spectrometry. The structure reveals a 36-nm particle in which the kinesin heavy chains (KHCs) adopt a head to tail configuration, stabilized by asymmetrically arranged kinesin light chain (KLC) tetratricopeptide repeat (TPR) domains that bind across folded KHC coiled-coils and wedge in between the KHC motor domains. This architecture inhibits kinesin motility by constraining the dimeric motor domains in a configuration that is incompatible with processive movement. In addition, the structure shows that the KLC C-terminal helices occlude the TPR cargo binding interfaces, revealing a second layer of autoinhibition that directly blocks cargo engagement. Functional studies and structural modeling suggest that binding of regulatory factors, such as MAP7D3, compete with intramolecular KHC coiled-coil interactions, resulting in the unfurling of the autoinhibited structure and activating motor motility. These findings provide a molecular framework for understanding kinesin-1 regulation and its implications for intracellular transport.

biochemistry↗

bioRxiv2025

Cryo-EM structures of the Plant Augmin reveal its intertwined coiled-coil assembly, antiparallel dimerization and NEDD1 binding mechanisms

Microtubule (MT) branch nucleation is fundamental for building parallel MT networks in eukaryotic cells. In plants and metazoans, MT branch nucleation requires Augmin and NEDD1 proteins which bind along MTs and then recruit and activate the gamma-tubulin ring complex ({gamma}-TuRC). Augmin is a fork-shaped assembly composed of eight coiled-coil subunits, while NEDD1 is a WD40 {beta}-propellor protein that bridges across MTs, Augmin, and {gamma}-TuRC during MT branch nucleation. Here, we reconstitute hetero-tetrameric and hetero-octameric Arabidopsis thaliana Augmin assemblies, resolve their subunit interactions using crosslinking mass spectrometry and determine 3.7 to 7.3-[A] cryo-EM structures for the V-junction and extended regions of Augmin. These structures allowed us to generate a complete de novo plant Augmin model that reveals the long-range multi coiled-coil interfaces that stabilize its 40-nm hetero-octameric fork-shaped organization. We discovered the dual calponin homology (CH) domain forming its MT binding site at the end of its V-junction undertake open and closed conformations. We determined a 12-[A] dimeric Augmin cryo-EM structure revealing Augmin undergoes anti-parallel dimerization through two conserved surfaces along Augmins extended region. We reconstituted the NEDD1 WD40 {beta}-propellor with Augmin revealing it directly binds on top its V-junction and enhances Augmin dimerization. Our studies suggest that cooperativity between the Augmin dual CH domains and NEDD1 WD40 binding site may regulate Augmin V-junction dual binding to MT lattices. This unique V-shaped dual binding and organization anchors Augmins along MTs generating a platform to recruit {gamma}-TuRC and activate branched MT nucleation.

biochemistry↗

bioRxiv2024

Cryo-EM structures of the tubulin cofactors reveal the molecular basis for the biogenesis of alpha/beta-tubulin

Microtubule polarity and dynamic polymerization originate from the self-association properties of the a-tubulin heterodimer. For decades, it has remained poorly understood how the tubulin cofactors, TBCD, TBCE, TBCC, and the Arl2 GTPase mediate a-tubulin biogenesis from - and {beta}-tubulins. Here, we use cryogenic electron microscopy to determine structures of tubulin cofactors bound to {beta}-tubulin. These structures show that TBCD, TBCE, and Arl2 form a heterotrimeric cage-like TBC-DEG assembly around the a-tubulin heterodimer. TBCD wraps around Arl2 and almost entirely encircles -tubulin, while TBCE forms a lever arm that anchors along the other end of TBCD and rotates -tubulin. Structures of the TBC-DEG-{beta}-tubulin assemblies bound to TBCC reveal the clockwise rotation of the TBCE lever that twists a-tubulin by pulling its C-terminal tail while TBCD holds -tubulin in place. Altogether, these structures uncover transition states in {beta}-tubulin biogenesis, suggesting a vise-like mechanism for the GTP-hydrolysis dependent a-tubulin biogenesis mediated by TBC-DEG and TBCC. These structures provide the first evidence of the critical functions of the tubulin cofactors as enzymes that regulate the invariant organization of {beta}-tubulin, by catalyzing - and {beta}-tubulin assembly, disassembly, and subunit exchange which are crucial for regulating the polymerization capacities of {beta}-tubulins into microtubules.

biochemistry↗

bioRxiv2021

The mechanochemical origins of the microtubule sliding motility within the kinesin-5 domain organization

The conserved kinesin-5 bipolar tetrameric motors slide apart microtubules during mitotic spindle assembly and elongation. Kinesin-5 bipolar organization originates from its conserved tetrameric helical minifilament, which position the C-terminal tail domains of two subunits near the N-terminal motor domains of two anti-parallel subunits (Scholey et al, 2014). This unique tetrameric structure enables kinesin-5 to simultaneously engage two microtubules and transmit forces between them, and for multiple kinesin-5 motors to organize via tail to motor interactions during microtubule sliding (Bodrug et al, 2020). Here, we show how these two structural adaptations, the kinesin-5 tail-motor domain interactions and the length of the tetrameric minifilament, determine critical aspects of kinesin-5 motility and sliding mechanisms. An x-ray structure of the 34-nm kinesin-5 minifilament reveals how the dual dimeric N-terminal coiled-coils emerge from the tetrameric central bundle. Using this structure, we generated active bipolar mini-tetrameric motors from Drosophila and human orthologs, which are half the length of native kinesin-5. Using single-molecule motility assays, we show that kinesin-5 tail domains promote mini-tetramers static pauses that punctuate processive motility. During such pauses, kinesin-5 mini-tetramers form multi-motor clusters mediated via tail to motor domain cross-interactions. These clusters undergo slow and highly processive motility and accumulate at microtubule plus-ends. In contrast to native kinesin-5, mini-tetramers require tail domains to initiate microtubule crosslinking. Although mini-tetramers are highly strained in initially aligning microtubules, they slide microtubules more efficiently than native kinesin-5, due to their decreased minifilament flexibility. Our studies reveal that the conserved kinesin-5 motor-tail mediated clustering and the length of the tetrameric minifilament are key features for sliding motility and are critical in organizing microtubules during mitotic spindle assembly and elongation.

cell biology↗

bioRxiv2020

Drag-induced directionality switching of kinesin-5 Cin8 revealed by cluster-motility analysis

Directed active motion of motor proteins is a vital process in virtually all eukaryotic cells. Nearly a decade ago, the discovery of directionality switching of mitotic kinesin-5 motors challenged the long-standing paradigm that individual kinesin motors are characterized by an intrinsic directionality. While several kinesin motors have now been shown to exhibit context-dependent directionality that can be altered under diverse experimental conditions, the underlying mechanism remains unknown. Here, we studied clustering-induced directionality switching of the mitotic kinesin-5 Cin8, using a fluorescence-based single-molecule motility assay combined with biophysical theory. Based on the detailed characterization of the motility of single motors and clusters of Cin8, we developed a predictive molecular model, that quantitatively agrees with experimental data. This combined approach allowed us to quantify the response of Cin8 motors to external forces as well as the interactions between Cin8 motors, and thereby develop a detailed understanding of the molecular mechanism underlying directionality switching. The main insight is that directionality switching is caused by a single feature of Cin8: an asymmetric response of active motion to forces that oppose motion, here referred to as drag. This general mechanism explains why bidirectional motor proteins are capable of reversing direction in response to seemingly unrelated experimental factors including clustering, changes in the ionic strength of the buffer, increased motor density and molecular crowding, and in motility assays. Significance StatementKinesin-5 motor proteins perform essential functions in chromosome segregation during mitotic cell division. Surprisingly, several kinesin-5 motors have the ability to reverse directionality under different experimental conditions, which contradicts the long-standing paradigm that individual kinesin motors are characterized by an intrinsic directionality. The mechanism underlying this ability to switch directionality has remained elusive. Here, we combine fluorescence-based motility assays and theoretical modeling to analyze cluster-size-dependent motility of the bidirectional kinesin-5 Cin8. Our results show that bidirectional motors can switch directionality because they exhibit an asymmetric response of active motion to drag. This mechanism explains multiple seemingly unrelated experimental factors that have been shown to cause directionality switching of kinesin motors.

biophysics↗

bioRxiv2019

The Kinesin-5 Tail Domain Directly Modulates the Mechanochemical Cycle of the Motor for Anti-Parallel Microtubule Sliding

Kinesin-5 motors organize mitotic spindles by sliding apart anti-parallel microtubules. They are homotetramers composed of two antiparallel dimers placing orthogonal motor and tail domains at opposite ends of a bipolar minifilament. Here, we describe a regulatory mechanism, involving direct binding of the tail to motor domain and reveal its fundamental role in microtubule sliding motility. Biochemical analyses reveal that the tail down-regulates microtubule-activated ATP hydrolysis by specifically engaging the motor in the nucleotide-free or ADP-bound states. Cryo-EM structures reveal that the tail stabilizes a unique conformation of the motor N-terminal subdomain opening its active site. Full-length kinesin-5 motors undergo slow motility and cluster together along microtubules, while tail-deleted motors exhibit rapid motility without clustering along microtubules. The tail is critical for motors to zipper together two microtubules by generating substantial forces within sliding zones. The tail domain is essential for kinesin-5 mitotic spindle localization in vivo, which becomes severely reduced when the tail is deleted. Our studies suggest a revised microtubule-sliding model, in which tail domains directly engage motor domains at both ends of kinesin-5 homotetramers enhancing stability of the dual microtubule-bound states leading to slow motility yet high force production.

biophysics↗

bioRxiv2018

Structural Basis of Tubulin Recruitment and Assembly by Tumor Overexpressed Gene (TOG) domain array Microtubule Polymerases

XMAP215/Stu2/Alp14 proteins accelerate microtubule plus-end polymerization by recruiting tubulins via arrays of Tumor Overexpressed Gene (TOG) domains. The underlying mechanism of these arrays as microtubule polymerases remains unknown. Here, we describe the biochemical and structural basis for TOG domain arrays in recruiting and polymerizing tubulins. Alp14 binds four tubulins via dimeric TOG1-TOG2 arrays, each with distinct exchange rates. X-ray structures reveal pseudo-dimeric square-shaped assemblies in which four TOG domains position four unpolymerized tubulins in a polarized wheel-like configuration. Crosslinking confirms square assemblies form in solution, and inactivation of their interfaces destabilizes square organizations without influencing tubulin binding. Using an approach to modulate tubulin polymerization, we determined a X-ray structure showing an unfurled assembly in which TOG1 and TOG2 uniquely bind two polymerized tubulins. Our findings suggest a new microtubule polymerase model in which TOG arrays recruit tubulins by forming square assemblies, which then unfurl facilitating their concerted polymerization into protofilaments.

biochemistry↗

bioRxiv2018

Roles for tubulin recruitment and self-organization by TOG domain arrays in Microtubule plus-end tracking and polymerase

The XMAP215/Stu2/Alp14 microtubule polymerases utilize Tumor Overexpressed Gene (TOG) domain arrays to accelerate microtubule plus-end polymerization. Structural studies suggest a microtubule polymerase model in which TOG arrays recruit four {beta}-tubulins, forming large square assemblies; an array of TOG1 and TOG2 domains may then unfurl from the square state to polymerize two {beta}-tubulins into protofilaments at microtubule ends. Here, we test this model using two biochemically characterized classes of fission yeast Alp14 mutants. Using in vitro reconstitution and in vivo live cell imaging, we show that {beta}-tubulins recruited by TOG1 and TOG2 domains serve non-additive roles in microtubule plus-end tracking and polymerase activities. Alp14 mutants with inactivated square assembly interfaces have defects in processive plus-end tracking and poor microtubule polymerase, indicating a functional role for square assemblies in processive tracking. These studies provide functional insights into how TOG1 and TOG2 domain arrays recruit tubulins and promote polymerase at microtubule plus ends.

biochemistry↗

bioRxiv2017

Structural Basis for Katanin Self-Assembly

The reorganization of microtubules in mitosis, meiosis and development requires the microtubule-severing activity of katanin. Katanin is composed of a AAA ATPase subunit and a regulatory subunit. Microtubule severing requires ATP hydrolysis by katanins conserved AAA ATPase domains. Whereas other AAA ATPases form stable hexamers, we show that wild-type katanin only forms heterodimers and heterotetramers. Heterododecamers were only observed for an ATP hydrolysis deficient mutant in the presence of ATP, suggesting an auto-inhibition mechanism that prevents oligomerization. X-ray structures of katanins AAA ATPase in monomeric nucleotide-free and pseudo-oligomeric ADP-bound states reveal conformational changes in AAA subdomains and N and C-terminal expansion segments that explain this auto-inhibition of assembly. These data lead to a model in which self-inhibited heterodimers bind to a microtubule, then transition into an assembly-competent conformation upon ATP binding. Microtubule-bound heterododecamers then promote tubulin extraction from the microtubule prior to oligomer dissociation.

biochemistry↗