RESUMEN
Isothermal titration calorimetry (ITC) is a widely used technique for the characterization of protein-protein and protein-ligand interactions. It provides information on the stoichiometry, affinity, and thermodynamic driving forces of interactions. This chapter exemplifies the use of ITC to investigate interactions between human autophagy modifiers (LC3/GABARAP proteins) and their interaction partners, the LIR motif-containing sequences. The purpose of this report is to present a detailed protocol for the production of LC3/GABARAP-interacting LIR peptides using E. coli expression systems. In addition, we outline the design of ITC experiments using the LC3/GABARAP:peptide interactions as an example. Comprehensive troubleshooting notes are provided to facilitate the adaptation of these protocols to different ligand-receptor systems. The methodology outlined for studying protein-ligand interactions will help to avoid common errors and misinterpretations of experimental results.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Proteínas Reguladoras de la Apoptosis , Calorimetría , Proteínas Asociadas a Microtúbulos , Unión Proteica , Termodinámica , Calorimetría/métodos , Humanos , Ligandos , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteínas Reguladoras de la Apoptosis/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Escherichia coli/metabolismo , Péptidos/química , Péptidos/metabolismoAsunto(s)
Microscopía por Crioelectrón , Microtúbulos , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Microtúbulos/química , Modelos Moleculares , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Humanos , Conformación Proteica , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestructuraRESUMEN
Microtubules are a major component of the cytoskeleton and can accumulate a plethora of modifications. The microtubule detyrosination cycle is one of these modifications; it involves the enzymatic removal of the C-terminal tyrosine of α-tubulin on assembled microtubules and the re-ligation of tyrosine on detyrosinated tubulin dimers. This modification cycle has been implicated in cardiac disease, neuronal development, and mitotic defects. The vasohibin and microtubule-associated tyrosine carboxypeptidase enzyme families are responsible for microtubule detyrosination. Their long-sought discovery allows to review and summarise differences and similarities between the two enzymes families and discuss how they interplay with other modifications and functions of the tubulin code.
Asunto(s)
Carboxipeptidasas , Microtúbulos , Tubulina (Proteína) , Tirosina , Microtúbulos/metabolismo , Humanos , Animales , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Carboxipeptidasas/metabolismo , Carboxipeptidasas/genética , Carboxipeptidasas/química , Tirosina/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/química , Procesamiento Proteico-PostraduccionalRESUMEN
Cosolvent molecular dynamics (MD) simulations have proven to be powerful in silico tools to predict hotspots for binding regions on protein surfaces. In the current study, the method was adapted and applied to two Tudor domain-containing proteins, namely Spindlin1 (SPIN1) and survival motor neuron protein (SMN). Tudor domains are characterized by so-called aromatic cages that recognize methylated lysine residues of protein targets. In the study, the conformational transitions from closed to open aromatic cage conformations were investigated by performing MD simulations with cosolvents using six different probe molecules. It is shown that a trajectory clustering approach in combination with volume and atomic distance tracking allows a reasonable discrimination between open and closed aromatic cage conformations and the docking of inhibitors yields very good reproducibility with crystal structures. Cosolvent MDs are suitable to capture the flexibility of aromatic cages and thus represent a promising tool for the optimization of inhibitors.
Asunto(s)
Simulación de Dinámica Molecular , Solventes , Solventes/química , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Fosfoproteínas/química , Dominios Proteicos , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Simulación del Acoplamiento Molecular , Conformación ProteicaRESUMEN
Rab6 is a key modulator of protein secretion. The dynein adapter Bicaudal D2 (BicD2) recruits the motors cytoplasmic dynein and kinesin-1 to Rab6GTP-positive vesicles for transport; however, it is unknown how BicD2 recognizes Rab6. Here, we establish a structural model for recognition of Rab6GTP by BicD2, using structure prediction and mutagenesis. The binding site of BicD2 spans two regions of Rab6 that undergo structural changes upon the transition from the GDP- to GTP-bound state, and several hydrophobic interface residues are rearranged, explaining the increased affinity of the active GTP-bound state. Mutations of Rab6GTP that abolish binding to BicD2 also result in reduced co-migration of Rab6GTP/BicD2 in cells, validating our model. These mutations also severely diminished the motility of Rab6-positive vesicles in cells, highlighting the importance of the Rab6GTP/BicD2 interaction for overall motility of the multi-motor complex that contains both kinesin-1 and dynein. Our results provide insights into trafficking of secretory and Golgi-derived vesicles and will help devise therapies for diseases caused by BicD2 mutations, which selectively affect the affinity to Rab6 and other cargoes.
Asunto(s)
Dineínas , Unión Proteica , Proteínas de Unión al GTP rab , Proteínas de Unión al GTP rab/metabolismo , Proteínas de Unión al GTP rab/genética , Humanos , Dineínas/metabolismo , Dineínas/química , Sitios de Unión , Cinesinas/metabolismo , Cinesinas/química , Cinesinas/genética , Mutación , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Transporte de Proteínas , Modelos Moleculares , Guanosina Trifosfato/metabolismoRESUMEN
Cep57, a vital centrosome-associated protein, recruits essential regulatory enzymes for centriole duplication. Its dysfunction leads to anomalies, including reduced centrioles and mosaic-variegated aneuploidy syndrome. Despite functional investigations, understanding structural aspects and their correlation with functions is partial till date. We present the structure of human Cep57 C-terminal microtubule binding (MT-BD) domain, revealing conserved motifs ensuring functional preservation across evolution. A leucine zipper, with an adjacent possible microtubule-binding region, potentially forms a stabilizing scaffold for microtubule nucleation-accommodating pulling and tension from growing microtubules. This study highlights conserved structural features of Cep57 protein, compares them with other analogous proteins, and explores how protein function is maintained across diverse organisms.
Asunto(s)
Proteínas de Ciclo Celular , Leucina Zippers , Microtúbulos , Unión Proteica , Humanos , Microtúbulos/metabolismo , Microtúbulos/química , Cristalografía por Rayos X , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Modelos Moleculares , Sitios de Unión , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Secuencia de Aminoácidos , Dominios Proteicos , Proteínas NuclearesRESUMEN
Microtubule (MT) filaments, composed of α/ß-tubulin dimers, are fundamental to cellular architecture, function and organismal development. They are nucleated from MT organizing centers by the evolutionarily conserved γ-tubulin ring complex (γTuRC). However, the molecular mechanism of nucleation remains elusive. Here we used cryo-electron tomography to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched budding yeast spindles. In our structure, γTuRC presents a ring of γ-tubulin subunits to seed nucleation of exclusively 13-protofilament MTs, adopting an active closed conformation to function as a perfect geometric template for MT nucleation. Our cryo-electron tomography reconstruction revealed that a coiled-coil protein staples the first row of α/ß-tubulin of the MT to alternating positions along the γ-tubulin ring of γTuRC. This positioning of α/ß-tubulin onto γTuRC suggests a role for the coiled-coil protein in augmenting γTuRC-mediated MT nucleation. Based on our results, we describe a molecular model for budding yeast γTuRC activation and MT nucleation.
Asunto(s)
Microscopía por Crioelectrón , Microtúbulos , Modelos Moleculares , Saccharomyces cerevisiae , Huso Acromático , Tubulina (Proteína) , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/química , Tubulina (Proteína)/ultraestructura , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Microtúbulos/química , Huso Acromático/metabolismo , Huso Acromático/ultraestructura , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestructura , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/ultraestructura , Tomografía con Microscopio Electrónico , Conformación Proteica , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/ultraestructuraRESUMEN
Microtubules are composed of α-tubulin and ß-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/ß-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.
Asunto(s)
Microscopía por Crioelectrón , Microtúbulos , Modelos Moleculares , Tubulina (Proteína) , Microtúbulos/metabolismo , Microtúbulos/química , Microtúbulos/ultraestructura , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Tubulina (Proteína)/ultraestructura , Humanos , Conformación Proteica , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/ultraestructuraRESUMEN
Cytoplasmic dynein is a microtubule motor vital for cellular organization and division. It functions as a ~4-megadalton complex containing its cofactor dynactin and a cargo-specific coiled-coil adaptor. However, how dynein and dynactin recognize diverse adaptors, how they interact with each other during complex formation, and the role of critical regulators such as lissencephaly-1 (LIS1) protein (LIS1) remain unclear. In this study, we determined the cryo-electron microscopy structure of dynein-dynactin on microtubules with LIS1 and the lysosomal adaptor JIP3. This structure reveals the molecular basis of interactions occurring during dynein activation. We show how JIP3 activates dynein despite its atypical architecture. Unexpectedly, LIS1 binds dynactin's p150 subunit, tethering it along the length of dynein. Our data suggest that LIS1 and p150 constrain dynein-dynactin to ensure efficient complex formation.
Asunto(s)
1-Alquil-2-acetilglicerofosfocolina Esterasa , Proteínas Adaptadoras Transductoras de Señales , Complejo Dinactina , Dineínas , Proteínas Asociadas a Microtúbulos , Proteínas del Tejido Nervioso , Microscopía por Crioelectrón , Complejo Dinactina/química , Complejo Dinactina/genética , Complejo Dinactina/metabolismo , Dineínas/química , Dineínas/genética , Dineínas/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/metabolismo , Microtúbulos/metabolismo , Unión Proteica , Humanos , Células HeLa , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Repeticiones WD40 , Mapeo de Interacción de ProteínasRESUMEN
Faithful chromosome segregation requires robust, load-bearing attachments of chromosomes to the mitotic spindle, a function accomplished by large macromolecular complexes termed kinetochores. In most eukaryotes, the constitutive centromere-associated network (CCAN) complex of the inner kinetochore recruits to centromeres the ten-subunit outer kinetochore KMN network that comprises the KNL1C, MIS12C and NDC80C complexes. The KMN network directly attaches CCAN to microtubules through MIS12C and NDC80C. Here, we determined a high-resolution cryo-EM structure of the human KMN network. This showed an intricate and extensive assembly of KMN subunits, with the central MIS12C forming rigid interfaces with NDC80C and KNL1C, augmented by multiple peptidic inter-subunit connections. We also observed that unphosphorylated MIS12C exists in an auto-inhibited state that suppresses its capacity to interact with CCAN. Ser100 and Ser109 of the N-terminal segment of the MIS12C subunit Dsn1, two key targets of Aurora B kinase, directly stabilize this auto-inhibition. Our study indicates how selectively relieving this auto-inhibition through Ser100 and Ser109 phosphorylation might restrict outer kinetochore assembly to functional centromeres during cell division.
Asunto(s)
Microscopía por Crioelectrón , Cinetocoros , Proteínas Asociadas a Microtúbulos , Modelos Moleculares , Proteínas Nucleares , Humanos , Cinetocoros/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Fosforilación , Aurora Quinasa B/metabolismo , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Conformación Proteica , Proteínas Cromosómicas no HistonaRESUMEN
Biorientation of chromosomes during cell division is necessary for precise dispatching of a mother cell's chromosomes into its two daughters. Kinetochores, large layered structures built on specialized chromosome loci named centromeres, promote biorientation by binding and sensing spindle microtubules. One of the outer layer main components is a ten-subunit assembly comprising Knl1C, Mis12C and Ndc80C (KMN) subcomplexes. The KMN is highly elongated and docks on kinetochores and microtubules through interfaces at its opposite extremes. Here, we combine cryogenic electron microscopy reconstructions and AlphaFold2 predictions to generate a model of the human KMN that reveals all intra-KMN interfaces. We identify and functionally validate two interaction interfaces that link Mis12C to Ndc80C and Knl1C. Through targeted interference experiments, we demonstrate that this mutual organization strongly stabilizes the KMN assembly. Our work thus reports a comprehensive structural and functional analysis of this part of the kinetochore microtubule-binding machinery and elucidates the path of connections from the chromatin-bound components to the force-generating components.
Asunto(s)
Microscopía por Crioelectrón , Cinetocoros , Proteínas Asociadas a Microtúbulos , Modelos Moleculares , Proteínas Nucleares , Humanos , Cinetocoros/metabolismo , Cinetocoros/ultraestructura , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas Asociadas a Microtúbulos/química , Proteínas Asociadas a Microtúbulos/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/química , Proteínas del Citoesqueleto/metabolismo , Proteínas del Citoesqueleto/química , Microtúbulos/metabolismo , Microtúbulos/ultraestructura , Unión Proteica , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/química , Células HeLaRESUMEN
Intellectual disabilities (ID) and autism spectrum disorders (ASD) have a variety of etiologies, including environmental and genetic factors. Our study reports a psychiatric clinical investigation and a molecular analysis using whole exome sequencing (WES) of two siblings with ID and ASD from a consanguineous family. Bioinformatic prediction and molecular docking analysis were also carried out. The two patients were diagnosed with profound intellectual disability, brain malformations such as cortical atrophy, acquired microcephaly, and autism level III. The neurological and neuropsychiatric examination revealed that P2 was more severely affected than P1, as he was unable to walk, presented with dysmorphic feature and exhibited self and hetero aggressive behaviors. The molecular investigations revealed a novel TRAPPC9 biallelic nonsense mutation (c.2920 C > T, p.R974X) in the two siblings. The more severely affected patient (P2) presented, along with the TRAPPC9 variant, a new missense mutation c.166 C > T (p.R56C) in the MID2 gene at hemizygous state, while his sister P1 was merely a carrier. The 3D modelling and molecular docking analysis revealed that c.166 C > T variant could affect the ability of MID2 binding to Astrin, leading to dysregulation of microtubule dynamics and causing morphological abnormalities in the brain. As our knowledge, the MID2 mutation (p.R56C) is the first one to be detected in Tunisia and causing phenotypic variability between the siblings. We extend the genetic and clinical spectrum of TRAPPC9 and MID2 mutations and highlights the possible concomitant presence of X-linked as well as autosomal recessive inheritance to causing ID, microcephaly, and autism.