Mechanical anisotropy of ankyrin repeats

Red blood cells are frequently deformed and their cytoskeletal proteins such as spectrin and ankyrin-R are repeatedly subjected to mechanical forces. While the mechanics of spectrin was thoroughly investigated in vitro and in vivo, little is known about the mechanical behavior of ankyrin-R. In this study, we combine coarse-grained steered molecular dynamics simulations and atomic force spectroscopy to examine the mechanical response of ankyrin repeats (ARs) in a model synthetic AR protein NI6C, and in the D34 fragment of native ankyrin-R when these proteins are subjected to various stretching geometry conditions. Our steered molecular dynamics results, supported by AFM measurements, reveal an unusual mechanical anisotropy of ARs: their mechanical stability is greater when their unfolding is forced to propagate from the N-terminus toward the C-terminus (repeats unfold at ~60 pN), as compared to the unfolding in the opposite direction (unfolding force ～ 30 pN). This anisotropy is also reflected in the complex refolding behavior of ARs. The origin of this unfolding and refolding anisotropy is in the various numbers of native contacts that are broken and formed at the interfaces between neighboring repeats depending on the unfolding/refolding propagation directions. Finally, we discuss how these complex mechanical properties of ARs in D34 may affect its behavior in vivo.     

Two C-terminal ankyrin repeats form the minimal stable unit of the ankyrin repeat protein p18INK4c

Ankyrin repeat proteins (ARPs) appear to be abundant in organisms from all phyla, and play critical regulatory roles, mediating specific interactions with target biomolecules and thus ordering the sequence of events in diverse cellular processes. ARPs possess a non-globular scaffold consisting of repeating motifs named ankyrin (ANK) repeats, which stack on each other. The modular architecture of ARPs provides a new paradigm for understanding protein stability and folding mechanisms. In the present study, the stability of various C-terminal fragments of the ARP p18^INK4c was investigated by all-atomic 450 ns molecular dynamics (MD) simulations in explicit water solvent. Only motifs with at least two ANK repeats made stable systems in the available timescale. All smaller fragments were unstable, readily losing their native fold and α-helical content. Since each non-terminal ANK repeat has two hydrophobic sides, we may hypothesize that at least one hydrophobic side must be fully covered and shielded from the water as a necessary, but not sufficient, condition to maintain ANK repeat stability. Consequently, at least two ANK repeats are required to make a stable ARP. Figure Structure of the p18INK4c protein (PDB entry 1IHB, chain B), which is a member of the cyclin-dependent kinase inhibitor (INK) tumor suppressor family with five ankyrin (ANK) repeat modules. The figure was generated by PyMol Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 1. Solution conformational and hydrodynamic properties.

To gain insight into the structural basis for Notch signaling, and to investigate the relationship between structure and stability in ankyrin repeat proteins, we have examined structural features of polypeptides from the Drosophila melanogaster Notch protein that contain five, six, and a putative seventh ankyrin repeat. Circular dichroism (CD) spectroscopy indicates that Notch ankyrin polypeptides of different length contain a significant amount of alpha-helix, indicating that a folded structure can be maintained despite the loss of individual ankyrin modules. However, the alpha-helical content of the construct with the putative seventh repeat is slightly higher than polypeptides containing fewer repeats, suggesting that the putative seventh repeat may help stabilize other parts of the ankyrin domain. Fluorescence spectroscopy indicates that the single tryptophan in the fifth ankyrin repeat is in a nonpolar environment and is shielded from solvent in all three constructs, although slight differences in spectroscopic properties of the six- and five-repeat constructs are observed, indicating minor structural changes. Near-UV CD indicates that these ankyrin polypeptides contain a significant amount of fixed tertiary structure surrounding their aromatic side chains. Gel filtration chromatography and sedimentation equilibrium studies indicate that these ankyrin repeat polypeptides are monomeric. Sedimentation velocity studies indicate that each polypeptide exhibits significant axial asymmetry, consistent with the elongated structure seen for other for ankyrin repeat proteins. However, the degree of asymmetry is greatest for the construct containing six repeats, suggesting that in the absence of the putative seventh repeat, the sixth repeat is partly unfolded.     

Sequential unfolding of ankyrin repeats in tumor suppressor p16

The ANK repeat is a ubiquitous 33-residue motif that adopts a beta hairpin helix-loop-helix fold. Multiple tandem repeats stack in a linear manner to produce an elongated structure that is stabilized predominantly by short-range interactions between residues close in sequence. The tumor suppressor p16(INK4) consists of four repeats and represents the minimal ANK folding unit. We found from Phi value analysis that p16 unfolded sequentially. The two N-terminal ANK repeats, which are distorted from the canonical ANK structure in all INK4 proteins and which are important for functional specificity, were mainly unstructured in the rate-limiting transition state for folding/unfolding, while the two C-terminal repeats were fully formed. A sequential unfolding mechanism could have implications for the cellular fate of wild-type and cancer-associated mutant p16 proteins.     

Multiple cadherin extracellular repeats mediate homophilic binding and adhesion.

The extracellular homophilic-binding domain of the cadherins consists of 5 cadherin repeats (EC1-EC5). Studies on cadherin specificity have implicated the NH(2)-terminal EC1 domain in the homophilic binding interaction, but the roles of the other extracellular cadherin (EC) domains have not been evaluated. We have undertaken a systematic analysis of the binding properties of the entire cadherin extracellular domain and the contributions of the other EC domains to homophilic binding. Lateral (cis) dimerization of the extracellular domain is thought to be required for adhesive function. Sedimentation analysis of the soluble extracellular segment of C-cadherin revealed that it exists in a monomer-dimer equilibrium with an affinity constant of approximately 64 microm. No higher order oligomers were detected, indicating that homophilic binding between cis-dimers is of significantly lower affinity. The homophilic binding properties of a series of deletion constructs, lacking successive or individual EC domains fused at the COOH terminus to an Fc domain, were analyzed using a bead aggregation assay and a cell attachment-based adhesion assay. A protein with only the first two NH(2)-terminal EC domains (CEC1-2Fc) exhibited very low activity compared with the entire extracellular domain (CEC1-5Fc), demonstrating that EC1 alone is not sufficient for effective homophilic binding. CEC1-3Fc exhibited high activity, but not as much as CEC1-4Fc or CEC1-5Fc. EC3 is not required for homophilic binding, however, since CEC1-2-4Fc and CEC1-2-4-5Fc exhibited high activity in both assays. These and experiments using additional EC combinations show that many, if not all, the EC domains contribute to the formation of the cadherin homophilic bond, and specific one-to-one interaction between particular EC domains may not be required. These conclusions are consistent with a previous study on direct molecular force measurements between cadherin ectodomains demonstrating multiple adhesive interactions (Sivasankar, S., W. Brieher, N. Lavrik, B. Gumbiner, and D. Leckband. 1999. PROC: Natl. Acad. Sci. USA. 96:11820-11824; Sivasankar, S., B. Gumbiner, and D. Leckband. 2001. Biophys J. 80:1758-68). We propose new models for how the cadherin extracellular repeats may contribute to adhesive specificity and function.     

Mutation of conserved histidines alters tertiary structure and nanomechanics of consensus ankyrin repeats

The conserved TPLH tetrapeptide motif of ankyrin repeats (ARs) plays an important role in stabilizing AR proteins, and histidine (TPLH)-to-arginine (TPLR) mutations in this motif have been associated with a hereditary human anemia, spherocytosis. Here, we used a combination of atomic force microscopy-based single-molecule force spectroscopy and molecular dynamics simulations to examine the mechanical effects of His → Arg substitutions in TPLH motifs in a model AR protein, NI6C. Our molecular dynamics results show that the mutant protein is less mechanically stable than the WT protein. Our atomic force microscopy results indicate that the mechanical energy input necessary to fully unfold the mutant protein is only half of that necessary to unfold the WT protein (53 versus 106 kcal/mol). In addition, the ability of the mutant to generate refolding forces is also reduced. Moreover, the mutant protein subjected to cyclic stretch-relax measurements displays mechanical fatigue, which is absent in the WT protein. Taken together, these results indicate that the His → Arg substitutions in TPLH motifs compromise mechanical properties of ARs and suggest that the origin of hereditary spherocytosis may be related to mechanical failure of ARs.     

Crystal structure of the ARF-GAP domain and ankyrin repeats of PYK2-associated protein beta

ADP ribosylation factors (ARFs), which are members of the Ras superfamily of GTP-binding proteins, are critical components of vesicular trafficking pathways in eukaryotes. Like Ras, ARFs are active in their GTP-bound form, and their duration of activity is controlled by GTPase-activating proteins (GAPs), which assist ARFs in hydrolyzing GTP to GDP. PAPbeta, a protein that binds to and is phosphorylated by the non-receptor tyrosine kinase PYK2, contains several modular signaling domains including a pleckstrin homology domain, an SH3 domain, ankyrin repeats and an ARF-GAP domain. Sequences of ARF-GAP domains show no recognizable similarity to those of other GAPs, and contain a characteristic Cys-X(2)-Cys-X(16-17)-Cys-X(2)-Cys motif. The crystal structure of the PAPbeta ARF-GAP domain and the C-terminal ankyrin repeats has been determined at 2.1 A resolution. The ARF-GAP domain comprises a central three-stranded beta-sheet flanked by five alpha-helices, with a Zn(2+) ion coordinated by the four cysteines of the cysteine-rich motif. Four ankyrin repeats are also present, the first two of which form an extensive interface with the ARF-GAP domain. An invariant arginine and several nearby hydrophobic residues are solvent exposed and are predicted to be the site of interaction with ARFs. Site-directed mutagenesis of these residues confirms their importance in ARF-GAP activity.     

A mechanical model of the gating spring mechanism of stereocilia

The stereocilium is the basic sensory unit of nature's mechanotransducers, which include the cochlear and vestibular organs. In noisy environments, stereocilia display high sensitivity to miniscule stimuli, effectively dealing with a situation that is a design challenge in micro systems. The gating spring hypothesis suggests that the mechanical stiffness of stereocilia bundle is softened by tip-link gating in combination with active bundle movement, contributing to the nonlinear amplification of miniscule stimuli. To demonstrate that the amplification is induced mechanically by the gating as hypothesized, we developed a biomimetic model of stereocilia and fabricated the model at the macro scale. The model consists of an inverted pendulum array with bistable buckled springs at its tips, which represent the mechanically gated ion channel. Model simulations showed that at the moment of gating, instantaneous stiffness softening generates an increase in response magnitude, which then sequentially occurs as the number of gating increases. This amplification mechanism appeared to be robust to the change of model parameters. Experimental data from the fabricated macro model also showed a significant increase in the open probability and pendulum deflection at the region having a smaller input magnitude. The results demonstrate that the nonlinear amplification of miniscule stimuli is mechanically produced by stiffness softening from channel gating.     

Insights into the roles of conserved and divergent residues in the ankyrin repeats of TRPV ion channels

Ion channels are often modulated by intracellular calcium levels. TRPV1, a channel responsible for the burning pain sensation in response to heat, acid or capsaicin, is desensitized at high intracellular calcium concentrations. We recently identified a multiligand-binding site in the N-terminal ankyrin repeat domain (ARD) of TRPV1 that binds ATP and sensitizes the channel. Calcium-calmodulin binds the same site and is necessary for calcium-mediated TRPV1 desensitization. Here, we examine in more detail the conservation of this TRPV1 multiligand-binding site in other species. Furthermore, using sequence analysis, we determine that the unusually twisted shape of the TRPV1-ARD is likely conserved in other TRPV channels, but not in the ARDs of other TRP subfamilies.     

Preparation and properties of human red-cell ankyrin.

We describe a procedure for the preparation of ankyrin from human red cells with a yield of 2-3 mg of protein from 30 ml of packed cells. This represents an improvement of an order of magnitude over the usual earlier procedure. Moreover, the product is, in our hands, much more stable against adsorption and proteolysis, and can in general be stored for at least 2 months at 4 degrees C without significant decrease in concentration and binding activity. The preparation depends on the release of the ankyrin-band-3 complex from the membrane cytoskeleton when intact cells are lysed in a medium containing concentrated Triton X-100. The complex is dissociated at high ionic strength, and the final purification is achieved by gel filtration in a medium containing 2 M-Tris or 0.6 M-NaBr. The ankyrin contains all the progression of components present in the intact membrane. All react with affinity-purified polyclonal anti-ankyrin antibodies, and all give widely similar patterns of peptides in partial proteolytic digests. The ankyrin is fully active, as judged by its capacity to bind to band-3-containing membrane vesicles and to Sepharose-coupled spectrin. All components bind to the membrane vesicles. Purified components 2.1 and 2.2, as well as the calmodulin-binding cytoskeletal constituent adducin, can be isolated in pure form by a single anion-exchange column step.     

Mutation in ankyrin repeats of the mouse Notch2 gene induces early embryonic lethality.

Notch family genes encode transmembrane proteins involved in cell-fate determination. Using gene targeting procedures, we disrupted the mouse Notch2 gene by replacing all but one of the ankyrin repeat sequences in the cytoplasmic domain with the E. coli (beta)-galactosidase gene. The mutant Notch2 gene encodes a 380 kDa Notch2-(beta)-gal fusion protein with (beta)-galactosidase activity. Notch2 homozygous mutant mice die prior to embryonic day 11.5, whereas heterozygotes show no apparent abnormalities and are fully viable. Analysis of Notch2 expression patterns, revealed by X-gal staining, demonstrated that the Notch2 gene is expressed in a wide variety of tissues including neuroepithelia, somites, optic vesicles, otic vesicles, and branchial arches, but not heart. Histological studies, including in situ nick end labeling procedures, showed earlier onset and higher incidence of apoptosis in homozygous mutant mice than in heterozygotes or wild type mice. Dying cells were particularly evident in neural tissues, where they were seen as early as embryonic day 9.5 in Notch2-deficient mice. Cells from Notch2 mutant mice attach and grow normally in culture, demonstrating that Notch2 deficiency does not interfere with cell proliferation and that expression of the Notch2-(beta)-gal fusion protein is not toxic per se. In contrast to Notch1-deficient mice, Notch2 mutant mice did not show disorganized somitogenesis, nor did they fail to properly regulate the expression of neurogenic genes such as Hes-5 or Mash1. In situ hybridization studies show no indication of altered Notch1 expression patterns in Notch2 mutant mice. The results indicate that Notch2 plays an essential role in postimplantation development in mice, probably in some aspect of cell specification and/or differentiation, and that the ankyrin repeats are indispensable for its function.