Structural Basis Underlying Strong Interactions between Ankyrins and Spectrins.

Ankyrins (encoded by ANK1/2/3 corresponding to Ankyrin-R/B/G or AnkR/B/G), via binding to spectrins, connect plasma membranes with actin cytoskeleton to maintain mechanical strengths and to modulate excitabilities of diverse cells such as neurons, muscle cells, and erythrocytes. Cellular and genetic evidences suggest that each isoform of ankyrins pairs with a specific ß-spectrin in discrete subcellular membrane microdomains for distinct functions, although the molecular mechanisms underlying such ankyrin/ß-spectrin pairings are unknown. In this study, we discover that a conserved and short extension N-terminal to the ZU5N-ZU5C-UPA tandem (exZZU) is critical for each ankyrin to bind to ß-spectrins with high affinities. Structures of AnkB/G exZZU in complex with spectrin repeats13-15 of ß2/ß4-spectrins solved here reveal that the extension sequence of exZZU forms an additional ß-strand contributing to the structural stability and enhanced affinity of each ZU5N/spectrin repeat interaction. The complex structures further reveal that the UPA domain of exZZU directly participates in spectrin binding. Formation of the exZZU supramodule juxtaposes the ZU5N and UPA domains for simultaneous interacting with spectrin repeats 14 and 15. However, our biochemical and structural investigations indicate that the direct and strong interactions between ankyrins and ß-spectrins do not appear to determine their pairing specificities. Therefore, there likely exists additional mechanism(s) for modulating functional pairings between ankyrins and ß-spectrins in cells.

Ultrastructure of the axonal periodic scaffold reveals a braid-like organization of actin rings.

Recent super-resolution microscopy studies have unveiled a periodic scaffold of actin rings regularly spaced by spectrins under the plasma membrane of axons. However, ultrastructural details are unknown, limiting a molecular and mechanistic understanding of these enigmatic structures. Here, we combine platinum-replica electron and optical super-resolution microscopy to investigate the cortical cytoskeleton of axons at the ultrastructural level. Immunogold labeling and correlative super-resolution/electron microscopy allow us to unambiguously resolve actin rings as braids made of two long, intertwined actin filaments connected by a dense mesh of aligned spectrins. This molecular arrangement contrasts with the currently assumed model of actin rings made of short, capped actin filaments. Along the proximal axon, we resolved the presence of phospho-myosin light chain and the scaffold connection with microtubules via ankyrin G. We propose that braided rings explain the observed stability of the actin-spectrin scaffold and ultimately participate in preserving the axon integrity.

Ankyrin repeats as a dimerization module.

Legionella pneumophila is a pathogen, causing severe pneumonia in humans called Legionnaires' disease. AnkC (LegA12) is a poorly characterized 495-residue effector protein conserved in multiple Legionella species. Here, we report the crystal structure of a C-terminally truncated AnkC (2-384) at 3.2 Å resolution. The structure shows seven ankyrin repeats (ARs) with unique structural features. AnkC forms a dimer along the outer surface of loops between ARs. The dimer exists both in the crystal form and in solution, as shown by analytical ultracentrifugation. This is the first example of ARs as a dimerization module as opposed to solely a protein interaction domain. In addition, a novel α-helix insert between AR3-AR4 is positioned across the surface opposite the ankyrin groove. Sequence conservation suggests that the ankyrin groove of AnkC is a functional site that interacts with binding targets. This ankyrin domain structure is an important step towards a functional characterization of AnkC.

Characterization of the axon initial segment of mice substantia nigra dopaminergic neurons.

The axon initial segment (AIS) is the site of initiation of action potentials and influences action potential waveform, firing pattern, and rate. In view of the fundamental aspects of motor function and behavior that depend on the firing of substantia nigra pars compacta (SNc) dopaminergic neurons, we identified and characterized their AIS in the mouse. Immunostaining for tyrosine hydroxylase (TH), sodium channels (Nav ) and ankyrin-G (Ank-G) was used to visualize the AIS of dopaminergic neurons. Reconstructions of sampled AIS of dopaminergic neurons revealed variable lengths (12-60 µm) and diameters (0.2-0.8 µm), and an average of 50% reduction in diameter between their widest and thinnest parts. Ultrastructural analysis revealed submembranous localization of Ank-G at nodes of Ranvier and AIS. Serial ultrathin section analysis and 3D reconstructions revealed that Ank-G colocalized with TH only at the AIS. Few cases of synaptic innervation of the AIS of dopaminergic neurons were observed. mRNA in situ hybridization of brain-specific Nav subunits revealed the expression of Nav 1.2 by most SNc neurons and a small proportion expressing Nav 1.6. The presence of sodium channels, along with the submembranous location of Ank-G is consistent with the role of AIS in action potential generation. Differences in the size of the AIS likely underlie differences in firing pattern, while the tapering diameter of AIS may define a trigger zone for action potentials. Finally, the conspicuous expression of Nav 1.2 by the majority of dopaminergic neurons may explain their high threshold for firing and their low discharge rate.

Structural and Energetic Characterization of the Ankyrin Repeat Protein Family.

Ankyrin repeat containing proteins are one of the most abundant solenoid folds. Usually implicated in specific protein-protein interactions, these proteins are readily amenable for design, with promising biotechnological and biomedical applications. Studying repeat protein families presents technical challenges due to the high sequence divergence among the repeating units. We developed and applied a systematic method to consistently identify and annotate the structural repetitions over the members of the complete Ankyrin Repeat Protein Family, with increased sensitivity over previous studies. We statistically characterized the number of repeats, the folding of the repeat-arrays, their structural variations, insertions and deletions. An energetic analysis of the local frustration patterns reveal the basic features underlying fold stability and its relation to the functional binding regions. We found a strong linear correlation between the conservation of the energetic features in the repeat arrays and their sequence variations, and discuss new insights into the organization and function of these ubiquitous proteins.

Caracterização molecular e funcional de ANKHD1 na hematopoese normal e neoplasica / Molecular and functional characterization of ANKHD1 in normal and neoplastic hematopoiesis

A identificação e caracterização estrutural e funcional de genes diferencialmente expressos entre tecidos tumorais e normais constituem etapas fundamentais para permitir a compreensão do processo neoplásico e o desenvolvimento de novas estratégias antitumorais. Ankyrin Repeat Single KH Domain containing 1 (ANKHD1) foi inicialmente identificada em células de adenocarcinoma de próstata humano (LNCaP), no ano de 2003. Entretanto, seu padrão de expressão e sua função ainda não haviam sido caracterizados. A ANKHD1 é uma proteína ortóloga à Multiple Ankyrin repeat and single KH domain (Mask) da Drosophila melanogaster. Mask foi identificada através de um rastreamento genético utilizado para detectar novas proteínas associadas à proteína tirosina fosfatase Corkscrew (CSW), homóloga à Src Homology-2 domain-containing protein tyrosine Phosphatase-2 (SHP2) humana. SHP2 é uma fosfatase de tirosina citoplasmática codificada pelo gene PTPN11 e exerce papel fundamental no desenvolvimento da hematopoese normal e leucêmica. Os objetivos gerais do presente estudo foram caracterizar o padrão de expressão gênica de ANKHD1 em células hematopoéticas normais e leucêmicas e verificar sua função nos processos celulares. Neste estudo foi demonstrado que o gene ANKHD1localiza-se no cromossomo 5, possui vários transcritos variantes possivelmente gerados por mecanismos de clivagem alternativa e codifica proteínas com domínios de repetições de anquirina. A região promotora desse gene possui vários elementos regulatórios importantes como...

The identification and the structural and functional characterization of genes differentially expressed between tumors and normal tissues are fundamental steps towards the understanding of the neoplastic process and the development of new anti-cancer strategies. The Ankyrin Repeat Single KH Domain containing 1 (ANKHD1) was first described in humans in a prostate carcinoma cell line LNCaP, in 2003; however, the expression pattern and function of ANKHD1 have not yet been described. ANKHD1 is an orthologous protein of the Drosophila melanogaster, MASK (Multiple Ankyrin repeat and single KH domain), where it was first identified using a genetic screen designed to discover proteins that interact with the protein tyrosine phosphatase Corkscrew (CSW), which is a homolog to the SH2-containing protein tyrosine phosphatase (SHP2) in humans. SHP2 is a cytoplasmic protein-tyrosine phosphatase, coded by the PTPN11 gene and plays an important role in the development of normal hematopoiese and leukemogenesis. The aim of the present study was to characterize the gene expression pattern of ANKHD1 in normal and leukemic hematopoietic cells and to determine their function in cellular process.This study has demonstrated that the ANKHD1 gene is located on chromosome 5, this gene has several possible variant transcripts...

Nanospring behaviour of ankyrin repeats.

Ankyrin repeats are an amino-acid motif believed to function in protein recognition; they are present in tandem copies in diverse proteins in nearly all phyla. Ankyrin repeats contain antiparallel alpha-helices that can stack to form a superhelical spiral. Visual inspection of the extrapolated structure of 24 ankyrin-R repeats indicates the possibility of spring-like behaviour of the putative superhelix. Moreover, stacks of 17-29 ankyrin repeats in the cytoplasmic domains of transient receptor potential (TRP) channels have been identified as candidates for a spring that gates mechanoreceptors in hair cells as well as in Drosophila bristles. Here we report that tandem ankyrin repeats exhibit tertiary-structure-based elasticity and behave as a linear and fully reversible spring in single-molecule measurements by atomic force microscopy. We also observe an unexpected ability of unfolded repeats to generate force during refolding, and report the first direct measurement of the refolding force of a protein domain. Thus, we show that one of the most common amino-acid motifs has spring properties that could be important in mechanotransduction and in the design of nanodevices.

The spectrin-ankyrin skeleton controls CD45 surface display and interleukin-2 production.

With T cell receptor stimulation, intracellular pools of CD45 and spectrin move to the surface. These processes are coupled. In both peripheral lymphocytes and Jurkat T cells, betaI spectrin and ankyrin associate with CD45. In Jurkat T cells, betaI spectrin peptides suppress surface recruitment of CD45 and CD3 and abrogate T cell activation. Other glycoproteins such as CD43 are not altered by the spectrin peptides. Spectrin's effects are mediated by ankyrin, which binds directly to the cytoplasmic domain of CD45 (K(d) = 4.3 +/- 3.0 nM). These data reveal a novel and unexpected contribution of the spectrin-ankyrin skeleton to the control of T lymphocyte function.