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At least 19 recordsLinked to original sources
PubMed2006

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.

Amino Acid Motifs↗
PubMed1993

The membrane-binding domain of ankyrin contains four independently folded subdomains, each comprised of six ankyrin repeats.

Ankyrin repeats are a 33-amino acid motif present in a number of proteins of diverse functions including transcription factors, cell differentiation molecules, and structural proteins. This motif has been shown to mediate protein interactions in the case of ankyrin as well as several other repeat-bearing proteins. In ankyrin, 24 tandemly arrayed repeats are arranged to form a globular, membrane-binding domain. This report provides evidence that the repeats in this domain fold into four independently folded subdomains of six repeats each. Limited proteolytic digestions of defined regions of the membrane-binding domain identified protease-sensitive sites, which divided this domain into subdomains of approximately six repeats each. Hydrodynamic measurements and circular dichroism spectroscopy of expressed subdomains confirmed that these six-repeat regions exist as folded, globular structures. The requirement of a complete set of six repeats for proper folding was determined using a series of protein constructs, which sequentially deleted repeats from the last subdomain. Deletion of even one repeat resulted in a 40% loss of alpha-helicity. Deletions removing three or more repeats abolished the helical signal completely. The spherical shapes of the intact domain and of the subdomains (inferred from hydrodynamic values) suggest that the four subdomains are organized in either a tetrahedral or square planar configuration. Two six-repeat subdomains were found to be required for high affinity association with the anion exchanger, suggesting that at least some of the protein interactions mediated by ankyrin repeats involve multiple subdomains.

Amino Acid Sequence↗
PubMed1998

Nuclear localization of IkappaB alpha is mediated by the second ankyrin repeat: the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences.

The ability of the IkappaB alpha protein to sequester dimeric NF-kappaB/Rel proteins in the cytoplasm provides an effective mechanism for regulating the potent transcriptional activation properties of NF-kappaB/Rel family members. IkappaB alpha can also act in the nucleus as a postinduction repressor of NF-kappaB/Rel proteins. The mechanism by which IkappaB alpha enters the nucleus is not known, as IkappaB alpha lacks a discernible classical nuclear localization sequence (NLS). We now report that nuclear localization of IkappaB alpha is mediated by a novel nuclear import sequence within the second ankyrin repeat. Deletion of the second ankyrin repeat or alanine substitution of hydrophobic residues within the second ankyrin repeat disrupts nuclear localization of IkappaB alpha. Furthermore, a region encompassing the second ankyrin repeat of IkappaB alpha is able to function as a discrete nuclear import sequence. The presence of a discrete nuclear import sequence in IkappaB alpha suggests that cytoplasmic sequestration of the NF-kappaB/Rel-IkappaB alpha complex is a consequence of the mutual masking of the NLS within NF-kappaB/Rel proteins and the import sequence within IkappaB alpha. Nuclear import may be a conserved property of ankyrin repeat domains (ARDs), as the ARDs from two other ARD-containing proteins, 53BP2 and GABPbeta, are also able to function as nuclear import sequences. We propose that the IkappaB alpha ankyrin repeats define a novel class of cis-acting nuclear import sequences.

Ankyrins↗
PubMed1998

The Elongin BC complex interacts with the conserved SOCS-box motif present in members of the SOCS, ras, WD-40 repeat, and ankyrin repeat families.

The Elongin BC complex was identified initially as a positive regulator of RNA polymerase II (Pol II) elongation factor Elongin A and subsequently as a component of the multiprotein von Hippel-Lindau (VHL) tumor suppressor complex, in which it participates in both tumor suppression and negative regulation of hypoxia-inducible genes. Elongin B is a ubiquitin-like protein, and Elongin C is a Skp1-like protein that binds to a BC-box motif that is present in both Elongin A and VHL and is distinct from the conserved F-box motif recognized by Skp1. In this report, we demonstrate that the Elongin BC complex also binds to a functional BC box present in the SOCS box, a sequence motif identified recently in the suppressor of cytokine signaling-1 (SOCS-1) protein, as well as in a collection of additional proteins belonging to the SOCS, ras, WD-40 repeat, SPRY domain, and ankyrin repeat families. In addition, we present evidence (1) that the Elongin BC complex is a component of a multiprotein SOCS-1 complex that attenuates Jak/STAT signaling by binding to Jak2 and inhibiting Jak2 kinase, and (2) that by interacting with the SOCS box, the Elongin BC complex can increase expression of the SOCS-1 protein by inhibiting its degradation. These results suggest that Elongin BC is a multifunctional regulatory complex capable of controlling multiple pathways in the cell through interaction with a short degenerate sequence motif found in many different proteins.

Amino Acid Sequence↗
PubMedDate not supplied

Altered expression of cardiac ankyrin repeat protein and its homologue, ankyrin repeat protein with PEST and proline-rich region, in atrophic muscles in amyotrophic lateral sclerosis.

OBJECTIVES: Cardiac ankyrin repeat protein, CARP, is a protein that is restrictedly expressed in the heart but barely expressed in skeletal muscles. Since CARP is induced by pressure overload to the heart, it is proposed to be a genetic marker for cardiac hypertrophy. We recently identified a novel protein, ankyrin repeat protein with PEST and proline-rich region (ARPP), which is homologous to CARP and is preferentially expressed in type 1 skeletal muscle fibers (cf. slow fibers). We also found that both ARPP and CARP expression is induced in experimentally denervated skeletal muscles in mice. Based on these findings, we hypothesized that their expression may be induced in skeletal muscles in neurodegenerating disease. This work aimed to determine the expression pattern of ARPP and CARP in amyotrophic lateral sclerosis (ALS). METHODS: In this study, we immunohistochemically analyzed the expression of ARPP and CARP in skeletal muscles of 9 ALS cases. RESULTS: We found that CARP was aberrantly expressed in atrophic skeletal muscle fibers in ALS. Although ARPP-positive fibers were randomly scattered in a checkerboard-like pattern in normal skeletal muscle, this pattern was absent in ALS muscles. Furthermore, we also found that ARPP was expressed in fast myosin heavy chain-positive fibers (cf. type 2 fiber). CONCLUSION: These findings suggest that type-specific expression patterns of ARPP and CARP are altered in skeletal muscles of ALS.

Aged↗
PubMed2000

ankA: an Ehrlichia phagocytophila group gene encoding a cytoplasmic protein antigen with ankyrin repeats.

Human granulocytic ehrlichiosis (HGE) is a potentially fatal, tick-borne disease caused by a bacterium related or identical to Ehrlichia phagocytophila. To identify and characterize E. phagocytophila group-specific protein antigen genes, we prepared and screened HGE agent and Ehrlichia equi genomic DNA expression libraries using polyclonal equine E. equi antibodies. Two clones, one each from HGE agent and E. equi, that were recognized specifically by antibodies to the E. phagocytophila group ehrlichiae had complete open reading frames of 3,693 and 3,615 nucleotides, respectively. The two clones were 96.6% identical and predicted a protein with at least 11 tandemly repeated ankyrin motifs. Thus, the gene was named ank (for ankyrin). When the encoded protein, named AnkA, was expressed in Escherichia coli, it was recognized by antibodies from rabbits and mice immunized with the HGE agent, sera from humans convalescent from HGE, and sera from horses convalescent from HGE and E. equi infection. Monospecific AnkA antibodies reacted with proteins in HGE agent immunoblots, and AnkA monoclonal antibodies detected cytoplasmic antigen in E. phagocytophila group bacteria and also detected antigen associated with chromatin in infected but not uninfected HL-60 cell cultures. These results suggest that this Ehrlichia protein may influence host cell gene expression.

Amino Acid Sequence↗
PubMed2005

Molecular cloning and characterization of a novel human gene containing 4 ankyrin repeat domains.

Ankyrin repeat, one of the most important protein motifs, plays a wide variety of roles in protein-protein interactions and in the signal pathways. Via large-scale sequencing, a novel 941-bp gene was isolated from an 18-week old human fetal brain cDNA library. It encodes a putative protein of 158 amino acid residues with four conserved ankyrin repeat domains. It displays a high degree of homology with rat low-density lipoprotein receptor-related protein 2-binding protein (Lrp2bp), and was therefore was named hLrp2bp (human Lrp2bp). The hLrp2bp gene was located in chromosome 4q35 and the conserved ankyrin repeat domains were located between amino acid residues 10 and 116. RT-PCR revealed that hLrp2bp was mainly expressed in the human testis, small intestine, colon and blood leukocytes, and in human pancreatic adenocarcinoma cells. A HEK293 cell was transfected with the ORF of hLrp2bp, and analyses showed that the protein was distributed both in the cytoplasm and nucleus.

Adaptor Proteins, Signal Transducing↗
PubMed2004

Folding of a designed simple ankyrin repeat protein.

Ankyrin repeats (AR) are 33-residue motifs containing a beta-turn, followed by two alpha-helices connected by a loop. AR occur in tandem arrangements and stack side-by-side to form elongated domains involved in very different cellular tasks. Recently, consensus libraries of AR repeats were constructed. Protein E1_5 represents a member of the shortest library, and consists of only a single consensus repeat flanked by designed N- and C-terminal capping repeats. Here we present a biophysical characterization of this AR domain. The protein is compactly folded, as judged from the heat capacity of the native state and from the specific unfolding enthalpy and entropy. From spectroscopic data, thermal and urea-induced unfolding can be modeled by a two-state transition. However, scanning calorimetry experiments reveal a deviation from the two-state behavior at elevated temperatures. Folding and unfolding at 5 degrees C both follow monoexponential kinetics with k(folding) = 28 sec(-1) and k(unfolding) = 0.9 sec(-1). Kinetic and equilibrium unfolding parameters at 5 degrees C agree very well. We conclude that E1_5 folds in a simple two-state manner at low temperatures while equilibrium intermediates become populated at higher temperatures. A chevron-plot analysis indicates that the protein traverses a very compact transition state along the folding/unfolding pathway. This work demonstrates that a designed minimal ankyrin repeat protein has the thermodynamic and kinetic properties of a compactly folded protein, and explains the favorable properties of the consensus framework.

Ankyrin Repeat↗
PubMed2002

Consensus-derived structural determinants of the ankyrin repeat motif.

The ankyrin repeat is one of the most common, modular, protein-protein interaction motifs in nature. To understand the structural determinants of this family of proteins and extract the consensus information that defines the architecture of this motif, we have designed a series of idealized ankyrin repeat proteins containing one, two, three, or four repeats by using statistical analysis of approximately 4,000 ankyrin repeat sequences from the PFAM database. Biophysical and x-ray crystallographic studies of the three and four repeat constructs (3ANK and 4ANK) to 1.26 and 1.5 A resolution, respectively, demonstrate that these proteins are well-folded, monomeric, display high thermostability, and adopt a very regular, tightly packed ankyrin repeat fold. Mapping the degree of amino acid conservation at each position on the 4ANK structure shows that most nonconserved residues are clustered on the surface of the molecule that has been designated as the binding site in naturally occurring ankyrin repeat proteins. Thus, the consensus amino acid sequence contains all information required to define the ankyrin repeat fold. Our results suggest that statistical analysis and the consensus sequence approach can be used as an effective method to design proteins with complex topologies. These generic ankyrin repeat proteins can serve as prototypes for dissecting the rules of molecular recognition mediated by ankyrin repeats and for engineering proteins with novel biological functions.

Amino Acid Sequence↗
PubMed2003

Designed to be stable: crystal structure of a consensus ankyrin repeat protein.

Ankyrin repeat (AR) proteins mediate innumerable protein-protein interactions in virtually all phyla. This finding suggested the use of AR proteins as designed binding molecules. Based on sequence and structural analyses, we designed a consensus AR with fixed framework and randomized interacting residues. We generated several combinatorial libraries of AR proteins consisting of defined numbers of this repeat. Randomly chosen library members are expressed in soluble form in the cytoplasm of Escherichia coli constituting up to 30% of total cellular protein and show high thermodynamic stability. We determined the crystal structure of one of those library members to 2.0-A resolution, providing insight into the consensus AR fold. Besides the highly complementary hydrophobic repeat-repeat interfaces and the absence of structural irregularities in the consensus AR protein, the regular and extended hydrogen bond networks in the beta-turn and loop regions are noteworthy. Furthermore, all residues found in the turn region of the Ramachandran plot are glycines. Many of these features also occur in natural AR proteins, but not in this rigorous and standardized fashion. We conclude that the AR domain fold is an intrinsically very stable and well-expressed scaffold, able to display randomized interacting residues. This scaffold represents an excellent basis for the design of novel binding molecules.

Amino Acid Sequence↗
PubMed2000

A minimum folding unit in the ankyrin repeat protein p16(INK4).

The ankyrin repeat is an abundant, 33 residue sequence motif that forms a consecutive beta-hairpin-helix-loop-helix (beta(2)alpha(2)) fold. Most ankyrin repeat proteins consist of four or more complete repeats, which provide stabilizing interactions between adjacent modules. The cyclin-dependent kinase inhibitor and tumor suppressor p16(INK4) (p16) is one of the smallest ankyrin repeat proteins with a known structure. It consists of four complete repeats plus short N and C-terminal flanking regions that are unstructured in solution. On the basis of preliminary proteolysis studies and predictions using a computer algorithm for identifying autonomous folding units, we have identified a fragment consisting of the third and fourth ankyrin repeats of p16, called p16C, that can fold independently, without the rest of the protein. Far-UV circular dichroism studies showed that p16C has a significant level of alpha-helical secondary structure, and two proline substitutions that disrupt the alpha-helical secondary structure in wild-type p16 disrupt the secondary structure in p16C. The thermal denaturation of p16C is cooperative and reversible, with a midpoint of transition at 30. 5(+/-1) degrees C. From urea-induced denaturation studies, the free energy of unfolding for p16C was estimated to be 1.7(+/-0.3) kcal/mol at 20 degrees C. (1)H-(15)N 2D NMR studies suggest that the ankyrin repeats in p16C are likely to fold into a structure similar to that of full-length p16. In order to define the minimum autonomous folding unit in p16, we have further dissected p16C into two complementary peptides, each containing a single ankyrin repeat. These peptides are unstructured in solution. Thus, p16C is the smallest ankyrin repeat module that is known to fold independently and, in general, we believe that the two-ankyrin repeat fold could be the minimum structural unit for all ankyrin repeat proteins. We further discuss the significance of p16C in protein folding and engineering.

Algorithms↗
PubMed1999

Expression and Functional Analysis of Yeast PHO81 Ankyrin Repeats.

There are six ankyrin repeats in yeast transcriptional factor PHO81. The PHO81 ankyrin repeats fused with glutathione S-transferase (GST-ANK) was overexpressed in E. coli and it existed in inclusion body form. Using reduced and oxidized glutathione systems the fused protein fragment was denatured and renatured and the soluble protein was obtained. Then it was purified by affinity purification through glutathione sepharose column and its activity was analyzed. The PHO85-PHO80 and PHO85-PAP1 kinase complexes were prepared via coimmunoprecipitation respectively. With purified recombinant PHO4 protein as substrate of the kinase complexes when the purified GST-ANK was added the kinase activity of PHO85-PHO80 or PHO85-PAP1 kinase complex was inhibited. These studies indicate that the ankyrin repeats interact with PHO85-PHO80 or PHO85-PAP1 complex inhibit their kinase activities and are crucial in interaction between PHO81 and other proteins.

Journal Article↗
PubMed2006

Ankyrin repeat: a unique motif mediating protein-protein interactions.

Ankyrin repeat, one of the most widely existing protein motifs in nature, consists of 30-34 amino acid residues and exclusively functions to mediate protein-protein interactions, some of which are directly involved in the development of human cancer and other diseases. Each ankyrin repeat exhibits a helix-turn-helix conformation, and strings of such tandem repeats are packed in a nearly linear array to form helix-turn-helix bundles with relatively flexible loops. The global structure of an ankyrin repeat protein is mainly stabilized by intra- and inter-repeat hydrophobic and hydrogen bonding interactions. The repetitive and elongated nature of ankyrin repeat proteins provides the molecular bases of the unique characteristics of ankyrin repeat proteins in protein stability, folding and unfolding, and binding specificity. Recent studies have demonstrated that ankyrin repeat proteins do not recognize specific sequences, and interacting residues are discontinuously dispersed into the whole molecules of both the ankyrin repeat protein and its partner. In addition, the availability of thousands of ankyrin repeat sequences has made it feasible to use rational design to modify the specificity and stability of physiologically important ankyrin repeat proteins and even to generate ankyrin repeat proteins with novel functions through combinatorial chemistry approaches.

Amino Acid Motifs↗
PubMed2004

Solution structure of the human oncogenic protein gankyrin containing seven ankyrin repeats and analysis of its structure--function relationship.

Human gankyrin (226 residues, 24.4 kDa) is a liver oncoprotein that plays an important role in the development of human hepatocellular carcinomas. In this paper, its solution structure is reported, which is the largest ankyrin protein ever determined by NMR. The highly degenerate primary sequences of the seven ankyrin repeats presented a major challenge, which was overcome by combined use of TROSY experiments, perdeuterated samples, isotope-filtered NMR experiments, and residual dipolar couplings. The final structure was of high quality, with atomic rmsds for the backbone (N, C', and C(alpha)) and all heavy atoms (residues 4-224) of 0.69 +/- 0.09 and 1.04 +/- 0.09 A, respectively. Detailed analyses of NMR data suggested that the conserved TPLH motifs play important structural roles in stabilizing the repeating ankyrin scaffold. Gankyrin is conformationally more stable than the tumor suppressor p16(INK4A), possibly due to the structural roles of conserved residues evidenced by slowly exchanging backbone amides as well as hydrogen bonding networks involving labile side chain protons. Structural comparison with p16(INK4A) identified several residues of gankyrin that are potentially important for CDK4 binding, whereas observation of the thiol proton of C180 indicated a well-structured Rb-binding site in the helical region of the sixth ankyrin repeat. Interestingly, the CDK4-binding site and Rb-binding site located in N- and C-terminal regions, respectively, are separated by comparatively more stable ankyrin repeats and highly condensed positive surface charge. These results and analyses will shed light on the structural basis of the function of human gankyrin.

Amino Acid Sequence↗
PubMed2006

Selection and characterization of Her2 binding-designed ankyrin repeat proteins.

Designed ankyrin repeat proteins (DARPins) are a novel class of binding proteins that bind their target protein with high affinity and specificity and have very favorable expression and stability properties. We describe here the in vitro selection of DARPins against human epidermal growth factor receptor 2 (Her2), an important target for cancer therapy and diagnosis. Several DARPins bind to the same epitope as trastuzumab (Herceptin), but none were selected that bind to the epitope of pertuzumab (Omnitarg). Some of the selected DARPins bind with low nanomolar affinity (Kd=7.3 nm) to the target. Further analysis revealed that all DARPins are highly specific and do not cross-react with epidermal growth factor receptor I (EGFR1) or any other investigated protein. The selected DARPins specifically bind to strongly Her2-overexpressing cell lines such as SKBR-3 but also recognize small amounts of Her2 on weakly expressing cell lines such as MCF-7. Furthermore, the DARPins also lead to a highly specific and strong staining of plasma membranes of paraffinated sections of human mamma-carcinoma tissue. Thus, the selected DARPins might be used for the development of diagnostic tests for the status of Her2 overexpression in different adenocarcinomas, and they may be further evaluated for their potential in targeted therapy since their favorable expression properties make the construction of fusion proteins very convenient.

Amino Acid Sequence↗
PubMed2004

The ankyrin repeat as molecular architecture for protein recognition.

The ankyrin repeat is one of the most frequently observed amino acid motifs in protein databases. This protein-protein interaction module is involved in a diverse set of cellular functions, and consequently, defects in ankyrin repeat proteins have been found in a number of human diseases. Recent biophysical, crystallographic, and NMR studies have been used to measure the stability and define the various topological features of this motif in an effort to understand the structural basis of ankyrin repeat-mediated protein-protein interactions. Characterization of the folding and assembly pathways suggests that ankyrin repeat domains generally undergo a two-state folding transition despite their modular structure. Also, the large number of available sequences has allowed the ankyrin repeat to be used as a template for consensus-based protein design. Such projects have been successful in revealing positions responsible for structure and function in the ankyrin repeat as well as creating a potential universal scaffold for molecular recognition.

Amino Acid Sequence↗
PubMed2006

Molecular dynamics study of the stabilities of consensus designed ankyrin repeat proteins.

Two designed ankyrin repeat (AR) proteins (E3_5 and E3_19) are high homologous (with about 87% sequence identity) and their crystal structures have a Calpha atom-positional root-mean-square difference of about 0.14 nm. However, it was found that E3_5 is considerably more stable than E3_19 in guanidinium hydrochloride and thermal denaturation experiments. With the goal of providing insights into the various factors contributing to the stabilities of the designed AR proteins and suggesting possible mutations to enhance their stabilities, homology modeling and molecular dynamics (MD) simulations with explicit solvent have been performed. Because the crystal structure of E3_19 was solved later than that of E3_5, a homology model of E3_19 based on the crystal structure of E3_5 was also used in the simulations. E3_5 shows a very stable trajectory in both crystal and solution simulations. In contrast, the C-terminal repeat of E3_19 unfolds in the simulations starting from either the modeled structure or the crystal structure, although it has a sequence identical to that of E3_5. A continuum electrostatic model was used to estimate the effect of single mutations on protein stability and to study the interaction between the internal ARs and the C-terminal capping AR. Mutations involving charged residues were found to have large effects on stability. Due to the difference in charge distribution in the internal ARs of E3_19 and E3_5, their interaction with the C-terminal capping AR is less favorable in E3_19. The simulation trajectories suggest that the stability of the designed AR proteins can be increased by optimizing the electrostatic interactions within and between the different repeats.

Ankyrin Repeat↗
PubMed2001

Analysis of ankyrin repeats reveals how a single point mutation in RFXANK results in bare lymphocyte syndrome.

Ankyrin repeats are well-known structural modules that mediate interactions between a wide spectrum of proteins. The regulatory factor X with ankyrin repeats (RFXANK) is a subunit of a tripartite RFX complex that assembles on promoters of major histocompatibility complex class II (MHC II) genes. Although it is known that RFXANK plays a central role in the nucleation of RFX, it was not clear how its ankyrin repeats mediate the interactions within the complex and with other proteins. To answer this question, we modeled the RFXANK protein and determined the variable residues of the ankyrin repeats that should contact other proteins. Site-directed alanine mutagenesis of these residues together with in vitro and in vivo binding studies elucidated how RFXAP and CIITA, which simultaneously interact with RFXANK in vivo, bind to two opposite faces of its ankyrin repeats. Moreover, the binding of RFXAP requires two separate surfaces on RFXANK. One of them, which is located in the ankyrin groove, is severely affected in the FZA patient with the bare lymphocyte syndrome. This genetic disease blocks the expression of MHC II molecules on the surface of B cells. By pinpointing the interacting residues of the ankyrin repeats of RFXANK, the mechanism of this subtype of severe combined immunodeficiency was revealed.

Amino Acid Sequence↗