Biology subjects

Leschziner, A. E.

Publications and source records attributed to Leschziner, A. E..

At least 19 recordsLinked to original sources

bioRxiv2026

Conformation governs cellular behaviors of LRRK2 Parkinson's disease variants

Gain-of-function of leucine-rich repeat kinase 2 (LRRK2) is a common cause of Parkinson's disease (PD). In its autoinhibited state, the leucine-rich repeats of LRRK2 block the kinase active site, preventing substrate phosphorylation. Displacing the repeats ('unlatching') primes LRRK2 for activity. Preferential unlatching causes increased substrate phosphorylation of the PD allele LRRK2[R1441H], but not LRRK2[G2019S]. Here, we define a large class of PD-associated 'unlatching' mutations based on their effect on LRRK2 conformation. Unlatching mutants show increased localization to membranes, the site of LRRK2's Rab substrates. Unlatching mutants are also characterized by decreased LRRK2 S935 phosphorylation, which we define as a biomarker of conformation. This framework resolves the mechanism of variants such as I2020T, which structural studies could not predict. Overall, we establish unlatching as a unifying mechanism for most PD-linked variants and describe measurable readouts for identifying LRRK2-driven disease.

cell biology↗

bioRxiv2026

Glk1p is an actin fold metabolic enzyme whose polymerization is sensitive to nucleotide state

The actin fold is present in enzymes ranging from sugar kinases to chaperones. Previous work on the actin fold metabolic enzyme, glucokinase (Glk1p) in S. cerevisiae found it could form filaments in response to its substrates, ATP and glucose (Stoddard et al., 2020). Here, we have identified the product, glucose 6-phosphate (G6P), as a second trigger for Glk1p polymerization in vitro. Furthermore, the addition of ADP to G6P-Glk1p filaments causes filament disassembly, suggesting that polymerization is sensitive to the state of the bound nucleotide and/or the transfer of the gamma phosphate. We have also identified a specific metabolic state, the accumulation of G6P during stationary phase, that triggers Glk1p polymerization in vivo. While the structures of Glk1p filaments in either the ATP/glucose or G6P-bound form are not similar to conventional actin filaments, the sensitivity of assembly to the gamma phosphate of the nucleotide provides a conceptual bridge between the cytoskeleton and metabolic regulation via enzyme polymerization. SummaryThe yeast glucokinase, Glk1p, is an actin fold enzyme that undergoes regulated polymerization in response to ATP/glucose and glucose 6-phosphate (G6P). Elevated G6P during stationary phase triggers Glk1p filament assembly in vivo consistent with a role in regulating glycolytic flux.

cell biology↗

bioRxiv2026

The structural basis for LRRK2's activation and autoinhibition

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are the second most common cause of autosomal-dominant Parkinsons disease (PD), and increased LRRK2 kinase activity is also observed in idiopathic PD, making LRRK2 a major actionable therapeutic target. LRRK2 is a 286-kDa multidomain enzyme containing a Ras-like GTPase (ROC) and a kinase domain. Using cryo-EM, biochemical reconstitution, and cell-based assays, we show that the ROC GTPase governs switching between autoinhibited and active states: GTP binding promotes activation, whereas GDP binding enforces autoinhibition. Two common PD-linked mutations, G2019S and R1441C/G/H, activate LRRK2 through distinct structural mechanisms, revealing genotype-specific routes to dysregulation. These findings provide a unified framework for understanding LRRK2 regulation with broad therapeutic implications. Stabilizing the GDP-bound state may inhibit LRRK2 by maintaining autoinhibition, whereas promoting the GTP-bound state could be advantageous in specific cellular contexts, such as the lung, where increased LRRK2 kinase activity may play protective or regulatory roles.

neuroscience↗

bioRxiv2025

In-cell cryo-electron tomography reveals differential effects of type I and type II kinase inhibitors on LRRK2 filament formation and microtubule association

Mutations in Leucine-Rich Repeat Kinase 2 (LRRK2) are a leading contributor to developing familial and idiopathic Parkinsons disease (PD). Most PD-causing LRRK2 mutations increase the kinase activity, leading to increased phosphorylation of Rab GTPases, disrupting vesicular trafficking, cytoskeletal dynamics, and autophagy. Under homeostatic conditions, the bulk of WT and PD-mutant LRRK2 is found in the cytosol. However, exogenously expressed LRRK2 can form microtubule-associated filaments that have been shown to affect molecular transport along microtubules in vitro. While the physiological relevance of microtubule binding has not been established yet, inhibitors being designed and tested as therapeutics have been shown to either promote or prevent filament formation of LRRK2. In this study, we examine the localization and resulting molecular organization of hyperactive LRRK2-I2020T, a common PD mutant, in cells treated with type I (MLi-2) or type II (GZD-824) kinase inhibitors. Treatment with a type I kinase inhibitor results in extensive LRRK2-I2020T decoration around microtubules and microtubule bundling. Stabilization of LRRK2-I2020T filaments by type I inhibitor treatment allowed us to build a full-length closed-kinase model of LRRK2-I2020T in its cellular environment. Conversely, treatment with a type II inhibitor resulted in minimal microtubule decoration by LRRK2-I2020T compared to Type I inhibitor treated cells. This study provides a structural framework for understanding how type I and type II kinase inhibitors differentially modulate LRRK2 filament formation, demonstrating that type I inhibitor treatment promotes a distinct filament architecture, whereas such assemblies are not observed with type II inhibitors.

biophysics↗

bioRxiv2025

Cryo-EM captures early intermediate steps in dynein activation by LIS1

Cytoplasmic dynein-1 (dynein) is an essential molecular motor in eukaryotic cells. Dynein primarily exists in an autoinhibited "Phi" state and requires conformational changes to assemble with its cofactors and form active transport complexes. LIS1, a key dynein regulator, enhances dynein activation and assembly. Using cryo-EM and a human dynein-LIS1 sample incubated with ATP, we mapped the conformational landscape of dynein activation by LIS1. We discovered a novel early intermediate state that we propose precedes our previously identified dynein-LIS1 "Chi" state. Mutations that disrupt this species, which we termed "Pre-Chi", led to motility defects in vitro, emphasizing its functional importance. Together, our findings provide new insights into how LIS1 relieves dynein autoinhibition during the activation pathway.

biophysics↗

bioRxiv2025

Structural basis of nucleosome recognition by the conserved Dsup and HMGN nucleosome-binding motif

The tardigrade damage suppressor (Dsup) and vertebrate high mobility group N (HMGN) proteins bind specifically to nucleosomes via a conserved motif whose structure has not been experimentally determined. Here we used cryo-EM to show that both proteins bind to the nucleosome acidic patch via analogous arginine anchors with one molecule bound to each face of the nucleosome. We additionally employed the natural promoter-containing 5S rDNA sequence for structural analysis of the nucleosome. These structures of an ancient nucle-osome-binding motif suggest that there is an untapped realm of proteins with a related mode of binding to chromatin.

molecular biology↗

bioRxiv2024

Type-II kinase inhibitors that target Parkinson's Disease-associated LRRK2

Aberrant increases in kinase activity of leucine-rich repeat kinase 2 (LRRK2) are associated with Parkinsons disease (PD). Numerous LRRK2-selective type-I kinase inhibitors have been developed and some have entered clinical trials. In this study, we present the first LRRK2-selective type-II kinase inhibitors. Targeting the inactive conformation of LRRK2 is functionally distinct from targeting the active-like conformation using type-I inhibitors. We designed these inhibitors using a combinatorial chemistry approach fusing selective LRRK2 type-I and promiscuous type-II inhibitors by iterative cycles of synthesis supported by structural biology and activity testing. Our current lead structures are selective and potent LRRK2 inhibitors. Through cellular assays, cryo-electron microscopy structural analysis, and in vitro motility assays, we show that our inhibitors stabilize the open, inactive kinase conformation. These new conformation-specific compounds will be invaluable as tools to study LRRK2s function and regulation, and expand the potential therapeutic options for PD.

cell biology↗

bioRxiv2024

Cryo-electron tomography reveals the microtubule-bound form of inactive LRRK2

Parkinsons Disease (PD) is the second most common neurodegenerative disorder. Mutations in leucine-rich repeat kinase 2 (LRRK2), a multi-domain protein containing both a kinase and a GTPase, are a leading cause of the familial form of PD. Pathogenic LRRK2 mutations increase LRRK2 kinase activity. While the bulk of LRRK2 is found in the cytosol, the protein associates with membranes where its Rab GTPase substrates are found, and under certain conditions, with microtubules. Integrative structural studies using single-particle cryo-electron microscopy (cryo-EM) and in situ cryo-electron tomography (cryo-ET) have revealed the architecture of microtubule-associated LRRK2 filaments, and that formation of these filaments requires LRRK2s kinase to be in the active-like conformation. However, whether LRRK2 can interact with and form filaments on microtubules in its autoinhibited state, where the kinase domain is in the inactive conformation and the N-terminal LRR domain covers the kinase active site, was not known. Using cryo-ET, we show that full-length LRRK2 can oligomerize on microtubules in its autoinhibited state. Both WT-LRRK2 and PD-linked LRRK2 mutants formed filaments on microtubules. While these filaments are stabilized by the same interfaces seen in the active-LRRK2 filaments, we observed a new interface involving the N-terminal repeats that were disordered in the active-LRRK2 filaments. The helical parameters of the autoinhibited-LRRK2 filaments are different from those reported for the active-LRRK2 filaments. Finally, the autoinhibited-LRRK2 filaments are shorter and less regular, suggesting they are less stable.

biophysics↗

bioRxiv2023

Development of LRRK2 designed ankyrin-repeat proteins

Leucine rich repeat kinase 2 (LRRK2) is a large multidomain protein containing two catalytic domains, a kinase and a GTPase, as well protein interactions domains, including a WD40 domain. The association of increased LRRK2 kinase activity with both the familial and sporadic forms of Parkinsons disease (PD) has led to intense interest in determining its cellular function. However, small molecule probes that can bind to LRRK2 and report on or affect its activity are needed. Here, we identified a series of high-affinity LRRK2-binding designed ankyrin-repeat proteins (DARPins). One of these DARPins (E11) bound to the LRRK2 WD40 domain with high affinity. LRRK2 bound to DARPin E11 showed improved behavior on cryo-EM grids, resulting in higher resolution LRRK2 structures. DARPin E11 did not affect the catalytic activity of a truncated form of LRRK2 in vitro but decreased the phosphorylation of Rab8A, a LRRK2 substrate, in cells. We also found that DARPin E11 disrupts the formation of microtubule-associated LRRK2 filaments in cells, which are known to require WD40-based dimerization. Thus, DARPin E11 is a new tool to explore the function and dysfunction of LRRK2 and guide the development of LRRK2 kinase inhibitors that target the WD40 domain instead of the kinase.

biochemistry↗

bioRxiv2023

Elf1 promotes Rad26 interaction with lesion-arrested Pol II for transcription-coupled repair

Transcription-coupled nucleotide excision repair (TC-NER) is a highly conserved DNA repair pathway that removes bulky lesions in the transcribed genome. Cockayne syndrome B protein (CSB), or its yeast ortholog Rad26, has been known for decades to play important roles in the lesion-recognition steps of TC-NER. Another conserved protein ELOF1, or its yeast ortholog Elf1, was recently identified as a core transcription-coupled repair factor. How Rad26 distinguishes between RNA polymerase II (Pol II) stalled at a DNA lesion or other obstacles and what role Elf1 plays in this process remains unknown. Here, we present cryo-EM structures of Pol II-Rad26 complexes stalled at different obstacles that show that Rad26 uses a universal mechanism to recognize a stalled Pol II but interacts more strongly with a lesion-arrested Pol II. A cryo-EM structure of lesion-arrested Pol II-Rad26 bound to Elf1 revealed that Elf1 induces new interactions between Rad26 and Pol II when the complex is stalled at a lesion. Biochemical and genetic data support the importance of the interplay between Elf1 and Rad26 in TC-NER initiation.

biophysics↗

bioRxiv2023

Inhibition of Parkinsons Disease-related LRRK2 by type-I and type-II kinase inhibitors: activity and structures

Mutations in Leucine Rich Repeat Kinase 2 (LRRK2) are a common cause of familial Parkinsons Disease (PD), and a risk factor for the sporadic form. Increased kinase activity has been shown in both familial and sporadic PD patients, making LRRK2 kinase inhibitors a major focus of drug development efforts in PD. Although significant progress has been made in understanding the structural biology of LRRK2, there are no available structures of LRRK2 inhibitor complexes. To this end, we solved cryo-EM structures of LRRK2, wild-type and PD-linked mutants, bound to the LRRK2-specific type-I inhibitor MLi-2 and the broad-spectrum type-II inhibitor GZD-824. Our structures revealed LRRK2s kinase in the active-like state, stabilized by type-I inhibitor interactions, and an inactive DYG-out type-II inhibitor complex. Our structural analysis also showed how inhibitor-induced conformational changes in LRRK2 are affected by its autoinhibitory N-terminal repeats. The structural models provide a template for the rational development of LRRK2 kinase inhibitors covering both canonical inhibitor binding modes.

biophysics↗

bioRxiv2022

Structure of LRRK1 and mechanisms of autoinhibition and activation

Leucine Rich Repeat Kinase 1 and 2 (LRRK1 and LRRK2) are homologs in the ROCO family of proteins in humans. Despite their shared domain architecture and involvement in intracellular trafficking, their disease associations are strikingly different: LRRK2 is involved in familial Parkinsons Disease (PD) while LRRK1 is linked to bone diseases. Furthermore, PD-linked mutations in LRRK2 are typically autosomal dominant gain-of-function while those in LRRK1 are autosomal recessive loss-of-function. To understand these differences, we solved cryo-EM structures of LRRK1 in its monomeric and dimeric forms. Both differ from the corresponding LRRK2 structures. Unlike LRRK2, which is sterically autoinhibited as a monomer, LRRK1 is sterically autoinhibited in a dimer-dependent manner. LRRK1 has an additional level of autoinhibition that prevents activation of the kinase and is absent in LRRK2. Finally, we place the structural signatures of LRRK1 and LRRK2 in the context of the evolution of the LRRK family of proteins.

biochemistry↗

bioRxiv2022

Lis1 relieves cytoplasmic dynein-1 auto-inhibition by acting as a molecular wedge

Cytoplasmic dynein-1 transports many intracellular cargos towards microtubule minus ends. Dynein is autoinhibited and undergoes conformational changes to form an active complex, consisting of one or two dynein dimers, the dynactin complex and activating adaptor(s)1,2. The Lissencephaly 1 gene, LIS1, is genetically linked to the dynein pathway from fungi to mammals and is mutated in patients with the neurodevelopmental disease lissencephaly3-5. Lis1 is required for active dynein complexes to form6-10, but how it does so is unclear. Here, we present a structure of two dynein motor domains with two Lis1 dimers wedged in-between. The contact sites between dynein and Lis1 in this structure, termed "Chi", are required for Lis1s regulation of dynein in Saccharomyces cerevisiae in vivo and the formation of active human dynein-dynactin- activating adaptor complexes in vitro. We propose that this structure represents an intermediate in dyneins activation pathway, revealing how Lis1 relieves dyneins autoinhibited state.

cell biology↗

bioRxiv2022

Structures of human cytoplasmic dynein in complex with the lissencephaly 1 protein, LIS1

The lissencephaly 1 protein, LIS1, is mutated in type-1 lissencephaly and is a key regulator of cytoplasmic dynein-1. At a molecular level, current models propose that LIS1 activates dynein by relieving its autoinhibited form. We recently reported a 3.1[A] structure of yeast dynein bound to Pac1, the yeast homologue of LIS1, which revealed the details of their interactions (Gillies et al., 2022). Based on this structure, we made mutations that disrupted these interactions and showed that they were required for dyneins function in vivo in yeast. We also used our yeast dynein-Pac1 structure to design mutations in human dynein to probe the role of LIS1 in promoting the assembly of active dynein complexes. These mutations had relatively mild effects on dynein activation, suggesting that there may be differences in how dynein and Pac1/LIS1 interact between yeast and humans. Here, we report cryo-EM structures of human dynein-LIS1 complexes. Our new structures reveal the differences between the yeast and human systems and provide a blueprint to disrupt the human dynein-LIS1 interaction more accurately. Our new structures also allow us to map type-1 lissencephaly disease mutations, as well as mutations in dynein linked to malformations of cortical development/ intellectual disability, in the context of the human dynein-LIS1 complex.

cell biology↗

bioRxiv2022

Structural basis for Parkinson's Disease-linked LRRK2's binding to microtubules

Leucine Rich Repeat Kinase 2 (LRRK2) is one of the most commonly mutated genes in familial Parkinsons Disease (PD). Under some circumstances, LRRK2 co-localizes with microtubules in cells, an association enhanced by PD mutations. We report a cryo-electron microscopy structure of the catalytic half of LRRK2, containing its kinase, which is in a closed conformation, and GTPase domains, bound to microtubules. We also report a structure of the catalytic half of LRRK1, which is closely related to LRRK2, but is not linked to PD. LRRK1s structure is similar to LRRK2, but LRRK1 does not interact with microtubules. Guided by these structures, we identify amino acids in LRRK2s GTPase domain that mediate microtubule binding; mutating them disrupts microtubule binding in vitro and in cells, without affecting LRRK2s kinase activity. Our results have implications for the design of therapeutic LRRK2 kinase inhibitors.

cell biology↗

bioRxiv2021

Multimodal tubulin binding by the yeast kinesin-8, Kip3, underlies its motility and depolymerization

The microtubule (MT) cytoskeleton is central to cellular processes including axonal growth, intracellular transport, and cell division, all of which rely on precise spatiotemporal control of MT organization. Kinesin-8s play a key role in regulating MT length by combining highly processive directional motility with MT-end disassembly. However, how kinesin-8 switches between these two apparently opposing activities remains unclear. Here, we define the structural features underlying this molecular switch through cryo-EM analysis of the yeast kinesin-8, Kip3 bound to MTs, and molecular dynamics simulations to approximate the complex of Kip3 with the curved tubulin state found at the MT plus-end. By integrating biochemical and single-molecule biophysical assays, we identified specific intra- and intermolecular interactions that modulate processive motility and MT disassembly. Our findings suggest that Kip3 undergoes conformational changes in response to tubulin curvature that underlie its unique ability to interact differently with the MT lattice than with the MT-end.

biophysics↗

bioRxiv2021

Structural Basis for Cytoplasmic Dynein-1 Regulation by Lis1

The lissencephaly 1 gene, LIS1, is mutated in patients with the neurodevelopmental disease lissencephaly. The Lis1 protein is conserved from fungi to mammals and is a key regulator of cytoplasmic dynein-1, the major minus-end-directed microtubule motor in many eukaryotes. Lis1 is the only dynein regulator that binds directly to dyneins motor domain, and by doing so alters dyneins mechanochemistry. Lis1 is required for the formation of fully active dynein complexes, which also contain essential cofactors: dynactin and an activating adaptor. Here, we report the first high-resolution structure of the yeast dynein-Lis1 complex. Our 3.1[A] structure reveals, in molecular detail, the major contacts between dynein and Lis1 and between Lis1s {beta}-propellers. Structure-guided mutations in Lis1 and dynein show that these contacts are required for Lis1s ability to form fully active human dynein complexes and to regulate yeast dyneins mechanochemistry and in vivo function. We present a model for the conserved role of Lis1 in regulating dynein from yeast to humans.

cell biology↗

bioRxiv2020

Structural insights into assembly and function of the RSC chromatin remodeling complex

Chromatin remodelers regulate the position and composition of nucleosomes throughout the genome, producing different remodeling outcomes despite a shared underlying mechanism based on a conserved RecA DNA translocase. How this functional diversity is achieved remains unknown despite recent cryo-electron microscopy (cryo-EM) reconstructions of several remodelers, including the yeast RSC complex. To address this, we have focused on a RSC subcomplex comprising its ATPase (Sth1), the essential actin-related proteins (ARPs) Arp7 and Arp9, and the fungal-specific protein Rtt102. Combining cryo-EM and biochemistry of this subcomplex, which exhibits regulation of remodeling by the ARPs, we show that ARP binding induces a helical conformation in the HSA domain of Sth1, which bridges the ATPase domain with the bulk of the complex. Surprisingly, the ARP module is rotated by 120{degrees} in the subcomplex relative to full RSC about a pivot point previously identified as a regulatory hub in Sth1, suggesting that large conformational changes are part of Sth1 regulation and RSC assembly. We also show that an interaction between Sth1 and the nucleosome acidic patch, which appears to be conserved among SWI/SNF remodelers, enhances remodeling. Taken together, our structural data shed light on the assembly and function of the RSC complex.

molecular biology↗