Structural analysis of microtubule binding by minus-end targeting protein Spiral2.

Microtubules (MTs) are dynamic cytoskeletal polymers that play a critical role in determining cell polarity and shape. In plant cells, acentrosomal MTs are localized on the cell surface and are referred to as cortical MTs. Cortical MTs nucleate in the cell cortex and detach from nucleation sites.

Crystal structure of the C-terminal domain of the plant-specific microtubule-associated protein Spiral2.

Plant cells form microtubule arrays, called `cortical microtubules', beneath the plasma membrane which are critical for cell-wall organization and directional cell growth. Cortical microtubules are nucleated independently of centrosomes. Spiral2 is a land-plant-specific microtubule minus-end-targeting protein that stabilizes the minus ends by inhibiting depolymerization of the filament.

The C-terminal region of the plasmid partitioning protein TubY is a tetramer that can bind membranes and DNA.

Bacterial low-copy-number plasmids require partition (par) systems to ensure their stable inheritance by daughter cells. In general, these systems consist of three components: a centromeric DNA sequence, a centromere-binding protein and a nucleotide hydrolase that polymerizes and functions as a motor. Type III systems, however, segregate plasmids using three proteins: the FtsZ/tubulin-like GTPase TubZ, the centromere-binding protein TubR and the MerR-like transcriptional regulator TubY.

CLASP2 binding to curved microtubule tips promotes flux and stabilizes kinetochore attachments.

CLASPs are conserved microtubule plus-end-tracking proteins that suppress microtubule catastrophes and independently localize to kinetochores during mitosis. Thus, CLASPs are ideally positioned to regulate kinetochore-microtubule dynamics required for chromosome segregation fidelity, but the underlying mechanism remains unknown. Here, we found that human CLASP2 exists predominantly as a monomer in solution, but it can self-associate through its C-terminal kinetochore-binding domain.

Cooperative DNA Binding of the Plasmid Partitioning Protein TubR from the Bacillus cereus pXO1 Plasmid.

Tubulin/FtsZ-like GTPase TubZ is responsible for maintaining the stability of pXO1-like plasmids in virulent Bacilli. TubZ forms a filament in a GTP-dependent manner, and like other partitioning systems of low-copy-number plasmids, it requires the centromere-binding protein TubR that connects the plasmid to the TubZ filament. Systems regulating TubZ partitioning have been identified in Clostridium prophages as well as virulent Bacillus species, in which TubZ facilitates partitioning by binding and towing the segrosome: the nucleoprotein complex composed of TubR and the centromere.

Molecular basis of the microtubule-regulating activity of microtubule crosslinking factor 1.

The variety of microtubule arrays observed across different cell types should require a diverse group of proteins that control microtubule organization. Nevertheless, mainly because of the intrinsic propensity of microtubules to easily form bundles upon stabilization, only a small number of microtubule crosslinking proteins have been identified, especially in postmitotic cells. Among them is microtubule crosslinking factor 1 (MTCL1) that not only interconnects microtubules via its N-terminal microtubule-binding domain (N-MTBD), but also stabilizes microtubules via its C-terminal microtubule-binding domain (C-MTBD).

CLASP2 Has Two Distinct TOG Domains That Contribute Differently to Microtubule Dynamics.

CLIP-associated proteins CLASPs are mammalian microtubule (MT) plus-end tracking proteins (+TIPs) that promote MT rescue in vivo. Their plus-end localization is dependent on other +TIPs, EB1 and CLIP-170, but in the leading edge of the cell, CLASPs display lattice-binding activity. MT association of CLASPs is suggested to be regulated by multiple TOG (tumor overexpressed gene) domains and by the serine-arginine (SR)-rich region, which contains binding sites for EB1.

MTCL1 crosslinks and stabilizes non-centrosomal microtubules on the Golgi membrane.

Recent studies have revealed the presence of a microtubule subpopulation called Golgi-derived microtubules that support Golgi ribbon formation, which is required for maintaining polarized cell migration. CLASPs and AKAP450/CG-NAP are involved in their formation, but the underlying molecular mechanisms remain unclear. Here, we find that the microtubule-crosslinking protein, MTCL1, is recruited to the Golgi membranes through interactions with CLASPs and AKAP450/CG-NAP, and promotes microtubule growth from the Golgi membrane.

CLASPs are required for proper microtubule localization of end-binding proteins.

Microtubule (MT) plus-end tracking proteins (+TIPs) preferentially localize to MT plus ends. End-binding proteins (EBs) are master regulators of the +TIP complex; however, it is unknown whether EBs are regulated by other +TIPs. Here, we show that cytoplasmic linker-associated proteins (CLASPs) modulate EB localization at MTs.

The novel PAR-1-binding protein MTCL1 has crucial roles in organizing microtubules in polarizing epithelial cells.

The establishment of epithelial polarity is tightly linked to the dramatic reorganization of microtubules (MTs) from a radial array to a vertical alignment of non-centrosomal MT bundles along the lateral membrane, and a meshwork under the apical and basal membranes. However, little is known about the underlying molecular mechanism of this polarity-dependent MT remodeling. The evolutionarily conserved cell polarity-regulating kinase PAR-1 (known as MARK in mammals), whose activity is essential for maintaining the dynamic state of MTs, has indispensable roles in promoting this process.

Crystallization and preliminary X-ray data analysis of the pXO1 plasmid-partitioning factor TubZ from Bacillus cereus.

TubZ is a structural homologue of tubulin and FtsZ GTPases, which are involved in the type III plasmid-partitioning system. TubZ assembles into polymers in a GTP-dependent manner and drives plasmid segregation as `cytomotive' filaments. In this study, C-terminally truncated TubZ from Bacillus cereus was crystallized in the presence or absence of GDP by the hanging-drop vapour-diffusion method.

Filament formation of the FtsZ/tubulin-like protein TubZ from the Bacillus cereus pXO1 plasmid.

Stable maintenance of low-copy-number plasmids requires partition (par) systems that consist of a nucleotide hydrolase, a DNA-binding protein, and a cis-acting DNA-binding site. The FtsZ/tubulin-like GTPase TubZ was identified as a partitioning factor of the virulence plasmids pBtoxis and pXO1 in Bacillus thuringiensis and Bacillus anthracis, respectively. TubZ exhibits high GTPase activity and assembles into polymers both in vivo and in vitro, and its "treadmilling" movement is required for plasmid stability in the cell.

A mutation of the fission yeast EB1 overcomes negative regulation by phosphorylation and stabilizes microtubules.

Mal3 is a fission yeast homolog of EB1, a plus-end tracking protein (+TIP). We have generated a mutation (89R) replacing glutamine with arginine in the calponin homology (CH) domain of Mal3. Analysis of the 89R mutant in vitro has revealed that the mutation confers a higher affinity to microtubules and enhances the intrinsic activity to promote the microtubule-assembly.

Characterization of a conserved "threonine clasp" in CAP-Gly domains: role of a functionally critical OH/pi interaction in protein recognition.

XH/pi hydrogen bonds have been predicted to make important contributions to protein structure and function. NMR evidence is presented for an OH/pi interaction between a highly conserved threonine and phenylalanine pair found specifically in CAP-Gly domains associated with mictrotubule plus ends. The functional contribution of this nonclassical hydrogen bond in target peptide recognition is demonstrated via subtle point mutagenesis.

CLIP170 autoinhibition mimics intermolecular interactions with p150Glued or EB1.

CLIP170 and p150(Glued) localize to the plus ends of growing microtubules. Using crystallography and NMR, we show that autoinhibitory interactions within CLIP170 use the same binding determinants as CLIP170's intermolecular interactions with p150(Glued). These interactions have both similar and distinct features when compared with the p150(Glued)-EB1 complex.

Crystallographic evidence for water-assisted photo-induced peptide cleavage in the stony coral fluorescent protein Kaede.

A coral fluorescent protein from Trachyphyllia geoffroyi, Kaede, possesses a tripeptide of His62-Tyr63-Gly64, which forms a chromophore with green fluorescence. This chromophore's fluorescence turns red following UV light irradiation. We have previously shown that such photoconversion is achieved by a formal beta-elimination reaction, which results in a cleavage of the peptide bond found between the amide nitrogen and the alpha-carbon at His62.

Structural basis for the activation of microtubule assembly by the EB1 and p150Glued complex.

Plus-end tracking proteins, such as EB1 and the dynein/dynactin complex, regulate microtubule dynamics. These proteins are thought to stabilize microtubules by forming a plus-end complex at microtubule growing ends with ill-defined mechanisms. Here we report the crystal structure of two plus-end complex components, the carboxy-terminal dimerization domain of EB1 and the microtubule binding (CAP-Gly) domain of the dynactin subunit p150Glued.

Crystal structure of the amino-terminal microtubule-binding domain of end-binding protein 1 (EB1).

The end-binding protein 1 (EB1) family is a highly conserved group of proteins that localizes to the plus-ends of microtubules. EB1 has been shown to play an important role in regulating microtubule dynamics and chromosome segregation, but its regulation mechanism is poorly understood. We have determined the 1.

The focal adhesion targeting (FAT) region of focal adhesion kinase is a four-helix bundle that binds paxillin.

Focal adhesion kinase (FAK) is a tyrosine kinase found in focal adhesions, intracellular signaling complexes that are formed following engagement of the extracellular matrix by integrins. The C-terminal 'focal adhesion targeting' (FAT) region is necessary and sufficient for localizing FAK to focal adhesions. We have determined the crystal structure of FAT and show that it forms a four-helix bundle that resembles those found in two other proteins involved in cell adhesion, alpha-catenin and vinculin.