Decoding microtubule detyrosination: enzyme families, structures, and functional implications.

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.

Molecular mechanism for recognition of the cargo adapter Rab6GTP by the dynein adapter BicD2.

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.

Exploring Aromatic Cage Flexibility Using Cosolvent Molecular Dynamics Simulations─An In-Silico Case Study of Tudor Domains.

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.

Structure of the γ-tubulin ring complex-capped microtubule.

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.

Structure of the native γ-tubulin ring complex capping spindle microtubules.

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.

Structure of the human outer kinetochore KMN network complex.

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.

Structure of the human KMN complex and implications for regulation of its assembly.

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.

Expanding the genetic and phenotypic spectrum of TRAPPC9 and MID2-related neurodevelopmental disabilities: report of two novel mutations, 3D-modelling, and molecular docking studies.

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.

Molecular mechanism of dynein-dynactin complex assembly by LIS1.

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.

Molecular Basis for SPINDOC-Spindlin1 Engagement and Its Role in Transcriptional Attenuation.

Spindlin1 is a histone reader with three Tudor-like domains and its transcriptional co-activator activity could be attenuated by SPINDOC. The first two Tudors are involved in histone methylation readout, while the function of Tudor 3 is largely unknown. Here our structural and binding studies revealed an engagement mode of SPINDOC-Spindlin1, in which a hydrophobic motif of SPINDOC, DOCpep3, stably interacts with Spindlin1 Tudor 3, and two neighboring K/R-rich motifs, DOCpep1 and DOCpep2, bind to the acidic surface of Spindlin1 Tudor 2. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPINDOC fragment containing the K/R-rich region attenuates histone or TCF4 binding by Spindlin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Further ChIP-seq and RT-qPCR indicated that SPINDOC could promote genomic relocation of Spindlin1, thus modulate downstream gene transcription. Collectively, we revealed multivalent engagement between SPINDOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPINDOC-Spindlin1 association, while K/R-rich region modulates the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activity of Spindlin1.

Structural mechanism of outer kinetochore Dam1-Ndc80 complex assembly on microtubules.

Kinetochores couple chromosomes to the mitotic spindle to segregate the genome during cell division. An error correction mechanism drives the turnover of kinetochore-microtubule attachments until biorientation is achieved. The structural basis for how kinetochore-mediated chromosome segregation is accomplished and regulated remains an outstanding question. In this work, we describe the cryo-electron microscopy structure of the budding yeast outer kinetochore Ndc80 and Dam1 ring complexes assembled onto microtubules. Complex assembly occurs through multiple interfaces, and a staple within Dam1 aids ring assembly. Perturbation of key interfaces suppresses yeast viability. Force-rupture assays indicated that this is a consequence of impaired kinetochore-microtubule attachment. The presence of error correction phosphorylation sites at Ndc80-Dam1 ring complex interfaces and the Dam1 staple explains how kinetochore-microtubule attachments are destabilized and reset.

Multivalent Molecular Tweezers Disrupt the Essential NDC80 Interaction with Microtubules.

Binding of microtubule filaments by the conserved Ndc80 protein is required for kinetochore-microtubule attachments in cells and the successful distribution of the genetic material during cell division. The reversible inhibition of microtubule binding is an important aspect of the physiological error correction process. Small molecule inhibitors of protein-protein interactions involving Ndc80 are therefore highly desirable, both for mechanistic studies of chromosome segregation and also for their potential therapeutic value. Here, we report on a novel strategy to develop rationally designed inhibitors of the Ndc80 Calponin-homology domain using Supramolecular Chemistry. With a multiple-click approach, lysine-specific molecular tweezers were assembled to form covalently fused dimers to pentamers with a different overall size and preorganization/stiffness. We identified two dimers and a trimer as efficient Ndc80 CH-domain binders and have shown that they disrupt the interaction between Ndc80 and microtubules at low micromolar concentrations without affecting microtubule dynamics. NMR spectroscopy allowed us to identify the biologically important lysine residues 160 and 204 as preferred tweezer interaction sites. Enhanced sampling molecular dynamics simulations provided a rationale for the binding mode of multivalent tweezers and the role of pre-organization and secondary interactions in targeting multiple lysine residues across a protein surface.

Effects of three microtubule-associated proteins (MAP2, MAP4, and Tau) on microtubules' physical properties and neurite morphology.

The physical properties of cytoskeletal microtubules have a multifaceted effect on the expression of their cellular functions. A superfamily of microtubule-associated proteins, MAP2, MAP4, and tau, promote the polymerization of microtubules, stabilize the formed microtubules, and affect the physical properties of microtubules. Here, we show differences in the effects of these three MAPs on the physical properties of microtubules. When microtubule-binding domain fragments of MAP2, tau, and three MAP4 isoforms were added to microtubules in vitro and observed by fluorescence microscopy, tau-bound microtubules showed a straighter morphology than the microtubules bound by MAP2 and the three MAP4 isoforms. Flexural rigidity was evaluated by the shape of the teardrop pattern formed when microtubules were placed in a hydrodynamic flow, revealing that tau-bound microtubules were the least flexible. When full-length MAPs fused with EGFP were expressed in human neuroblastoma (SH-SY5Y) cells, the microtubules in apical regions of protrusions expressing tau were straighter than in cells expressing MAP2 and MAP4. On the other hand, the protrusions of tau-expressing cells had the fewest branches. These results suggest that the properties of microtubules, which are regulated by MAPs, contribute to the morphogenesis of neurites.

The Roles of Microtubule-Associated Protein 4 in Wound Healing and Human Diseases.

Microtubules (MTs) are essential structural elements of cells. MT stability and dynamics play key roles in integrity of cell morphology and various cellular activities. The MT-associated proteins (MAPs) are specialized proteins that interact with MT and induce MT assemble into distinct arrays. Microtubule-associated protein 4 (MAP4), a member of MAPs family, ubiquitously expressed in both neuronal and non-neuronal cells and tissues, plays a key role in regulating MT stability. Over the past 40 years or so, the mechanism of MAP4 regulating MT stability has been well studied. In recent years, more and more studies have found that MAP4 affects the activities of sundry human cells through regulating MT stability with different signaling pathways, plays important roles in the pathogenesis of a number of disorders. The aim of this review is to outline the detailed regulatory mechanisms of MAP4 in MT stability, and to focus on its specific mechanisms in wound healing and various human diseases, thus to highlight the possibility of MAP4 as a future therapeutic target for accelerating wound healing and treating other disorders.

Resonance assignments of the microtubule-binding domain of the microtubule-associated protein 7 (MAP7).

The microtubule-associated protein 7 (MAP7) is a protein involved in cargo transport along microtubules (MTs) by interacting with kinesin-1 through the C-terminal kinesin-binding domain. Moreover, the protein is reported to stabilize MT, thereby playing a key role in axonal branch development. An important element for this latter function is the 112 amino-acid long N-terminal microtubule-binding domain (MTBD) of MAP7. Here we report NMR backbone and side-chain assignments that suggest a primarily alpha-helical secondary fold of this MTBD in solution. The MTBD contains a central long α-helical segment that includes a short four-residue 'hinge' sequence with decreased helicity and increased flexibility. Our data represent a first step towards analysing the complex interaction of MAP7 with MTs at an atomic level via NMR spectroscopy.

CryoEM shows the active dynein complex on microtubules.

In a recent study, Chaaban and Carter use cryo-electron microscopy (cryo-EM) and an innovative data-processing pipeline to determine the first high-resolution structure of the dynein-dynactin-BICDR1 complex assembled on microtubules. The structure of the complex reveals novel stoichiometry and provides new mechanistic insight into dynein function and mechanism.

The augmin complex architecture reveals structural insights into microtubule branching.

In mitosis, the augmin complex binds to spindle microtubules to recruit the γ-tubulin ring complex (γ-TuRC), the principal microtubule nucleator, for the formation of branched microtubules. Our understanding of augmin-mediated microtubule branching is hampered by the lack of structural information on the augmin complex. Here, we elucidate the molecular architecture and conformational plasticity of the augmin complex using an integrative structural biology approach. The elongated structure of the augmin complex is characterised by extensive coiled-coil segments and comprises two structural elements with distinct but complementary functions in γ-TuRC and microtubule binding, linked by a flexible hinge. The augmin complex is recruited to microtubules via a composite microtubule binding site comprising a positively charged unordered extension and two calponin homology domains. Our study provides the structural basis for augmin function in branched microtubule formation, decisively fostering our understanding of spindle formation in mitosis.

Structure-Based Design of Stapled Peptides That Bind GABARAP and Inhibit Autophagy.

The LC3/GABARAP family of proteins is involved in nearly every stage of autophagy. Inhibition of LC3/GABARAP proteins is a promising approach to blocking autophagy, which sensitizes advanced cancers to DNA-damaging chemotherapy. Here, we report the structure-based design of stapled peptides that inhibit GABARAP with nanomolar affinities. Small changes in staple structure produced stapled peptides with very different binding modes and functional differences in LC3/GABARAP paralog selectivity, ranging from highly GABARAP-specific to broad inhibition of both subfamilies. The stapled peptides exhibited considerable cytosolic penetration and resistance to biological degradation. They also reduced autophagic flux in cultured ovarian cancer cells and sensitized ovarian cancer cells to cisplatin. These small, potent stapled peptides represent promising autophagy-modulating compounds that can be developed as novel cancer therapeutics and novel mediators of targeted protein degradation.

Posttranslational modification of microtubules by the MATCAP detyrosinase.

The detyrosination-tyrosination cycle involves the removal and religation of the C-terminal tyrosine of α-tubulin and is implicated in cognitive, cardiac, and mitotic defects. The vasohibin-small vasohibin-binding protein (SVBP) complex underlies much, but not all, detyrosination. We used haploid genetic screens to identify an unannotated protein, microtubule associated tyrosine carboxypeptidase (MATCAP), as a remaining detyrosinating enzyme. X-ray crystallography and cryo-electron microscopy structures established MATCAP's cleaving mechanism, substrate specificity, and microtubule recognition. Paradoxically, whereas abrogation of tyrosine religation is lethal in mice, codeletion of MATCAP and SVBP is not. Although viable, defective detyrosination caused microcephaly, associated with proliferative defects during neurogenesis, and abnormal behavior. Thus, MATCAP is a missing component of the detyrosination-tyrosination cycle, revealing the importance of this modification in brain formation.