Studies of the ankyrin repeats of the Drosophila melanogaster Notch receptor. 2. Solution stability and cooperativity of unfolding.

To define the boundaries of the Drosophila Notch ankyrin domain, examine the effects of repeat number on the folding of this domain, and examine the degree to which the modular architecture of ankyrin repeat proteins results in modular stability, we have investigated the thermodynamics of unfolding of polypeptides corresponding to different segments of the ankyrin repeats of Drosophila Notch. We find that a polypeptide containing the six previously identified ankyrin repeats unfolds cooperatively, but is of modest stability. However, inclusion of a putative seventh, C-terminal ankyrin sequence doubles the stability of the Notch ankyrin domain (a 1000-fold increase in the folding equilibrium constant), indicating that the seventh ankyrin repeat is an important part of the Notch ankyrin domain, and demonstrating long-range interactions among ankyrin repeats. This putative seven-repeat polypeptide also shows increases in enthalpy, denaturant dependence (m-value), and heat capacity of unfolding (DeltaC(p)()) of around 50% each, suggesting that deletion of the seventh repeat results in partial unfolding of the sixth ankyrin repeat, consistent with spectroscopic and hydrodynamic data reported in the preceding paper [Zweifel, M. E., and Barrick, D. (2001) Biochemistry 40, 14344-14356]. A polypeptide consisting of only the five N-terminal repeats has stability similar to the six-repeat construct, demonstrating that stability is distributed asymmetrically along the ankyrin domain. These data are consistent with highly cooperative two-state folding of these ankyrin polypeptides, despite their modular architecture.     

Structure and stability of the ankyrin domain of the Drosophila Notch receptor

The Notch receptor contains a conserved ankyrin repeat domain that is required for Notch-mediated signal transduction. The ankyrin domain of Drosophila Notch contains six ankyrin sequence repeats previously identified as closely matching the ankyrin repeat consensus sequence, and a putative seventh C-terminal sequence repeat that exhibits lower similarity to the consensus sequence. To better understand the role of the Notch ankyrin domain in Notch-mediated signaling and to examine how structure is distributed among the seven ankyrin sequence repeats, we have determined the crystal structure of this domain to 2.0 angstroms resolution. The seventh, C-terminal, ankyrin sequence repeat adopts a regular ankyrin fold, but the first, N-terminal ankyrin repeat, which contains a 15-residue insertion, appears to be largely disordered. The structure reveals a substantial interface between ankyrin polypeptides, showing a high degree of shape and charge complementarity, which may be related to homotypic interactions suggested from indirect studies. However, the Notch ankyrin domain remains largely monomeric in solution, demonstrating that this interface alone is not sufficient to promote tight association. Using the structure, we have classified reported mutations within the Notch ankyrin domain that are known to disrupt signaling into those that affect buried residues and those restricted to surface residues. We show that the buried substitutions greatly decrease protein stability, whereas the surface substitutions have only a marginal affect on stability. The surface substitutions are thus likely to interfere with Notch signaling by disrupting specific Notch-effector interactions and map the sites of these interactions.     

The notch ankyrin domain folds via a discrete, centralized pathway

The Notch ankyrin repeat domain contains seven ankyrin sequence repeats, six of which adopt very similar structures. To determine if folding proceeds along parallel pathways and the order in which repeats become structured during folding, we examined the effect of analogous destabilizing Ala-->Gly substitutions in each repeat on folding kinetics. We find that folding proceeds to an on-pathway kinetic intermediate through a transition state ensemble containing structure in repeats three through five. Repeats two, six, and seven remain largely unstructured in this intermediate, becoming structured in a second kinetic step that leads to the native state. These data suggest that the Notch ankyrin domain folds according to a discrete kinetic pathway despite structural redundancy in the native state and highlight the importance of sequence-specific interactions in controlling pathway selection. This centralized pathway roughly corresponds to a low energy channel through the experimentally determined energy landscape.     

Structural basis for recognition of 2',5'-linked oligoadenylates by human ribonuclease L

An interferon-induced endoribonuclease, ribonuclease L (RNase L), is implicated in both the molecular mechanism of action of interferon and the fundamental control of RNA stability in mammalian cells. RNase L is catalytically active only after binding to an unusual activator molecule containing a 5'-phosphorylated 2',5'-linked oligoadenylate (2-5A), in the N-terminal half. Here, we report the crystal structure of the N-terminal ankyrin repeat domain (ANK) of human RNase L complexed with the activator 2-5A. This is the first structural view of an ankyrin repeat structure directly interacting with a nucleic acid, rather than with a protein. The ANK domain folds into eight ankyrin repeat elements and forms an extended curved structure with a concave surface. The 2-5A molecule is accommodated at a concave site and directly interacts with ankyrin repeats 2-4. Interestingly, two structurally equivalent 2-5A binding motifs are found at repeats 2 and 4. The structural basis for 2-5A recognition by ANK is essential for designing stable 2-5As with a high likelihood of activating RNase L.     

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.     

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.     

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.     

Folding mechanism of an ankyrin repeat protein: scaffold and active site formation of human CDK inhibitor p19(INK4d)

The p19(INK4d) protein consists of five ankyrin repeats (ANK) and controls the human cell cycle by inhibiting the cyclin D-dependent kinases (CDK) 4 and 6. We investigated the folding of p19(INK4d) by urea-induced unfolding transitions, kinetic analyses of unfolding and refolding, including double-mixing experiments and a special assay for folding intermediates. Folding is a sequential two-step reaction via a hyperfluorescent on-pathway intermediate. This intermediate is present under all conditions, during unfolding, refolding and at equilibrium. The folding mechanism was confirmed by a quantitative global fit of a consistent set of equilibrium and kinetic data revealing the thermodynamics and intrinsic folding rates of the different states. Surprisingly, the N<-->I transition is much faster compared to the I<-->U transition. The urea-dependence of the intrinsic folding rates causes population of the intermediate at equilibrium close to the transition midpoint. NMR detected hydrogen/deuterium exchange and the analysis of truncated variants showed that the C-terminal repeats ANK3-5 are already folded in the on-pathway intermediate, whereas the N-terminal repeats 1 and 2 are not folded. We suggest that during refolding, repeats ANK3-ANK5 first form the scaffold for the subsequent assembly of repeats ANK1 and ANK2. The binding function of p19(INK4d) resides in the latter repeats. We propose that the graded stability and the facile unfolding of repeats 1 and 2 is a prerequisite for the down-regulation of the inhibitory activity of p19(INK4d) during the cell-cycle.     

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies.     

Enhancing the stability and folding rate of a repeat protein through the addition of consensus repeats

Repeat proteins are constructed from a linear array of modular units, giving rise to an overall topology lacking long-range interactions. This suggests that stabilizing repeat modules based on consensus information might be added to a repeat protein domain, allowing it to be extended without altering its overall topology. Here we add consensus modules the ankyrin repeat domain from the Drosophila Notch receptor to investigate the structural tolerance to these modules, the relative thermodynamic stability of these hybrid proteins, and how alterations in the energy landscape influence folding kinetics. Insertions of consensus modules between repeats five and six of the Notch ankyrin domain have little effect on the far and near-UV CD spectra, indicating that neither secondary nor tertiary structure is dramatically altered. Furthermore, stable structure is maintained at increased denaturant concentrations in the polypeptides containing the consensus repeats, indicating that the consensus modules are capable of stabilizing much of the domain. However, insertion of the consensus repeats appears to disrupt cooperativity, producing a two-stage (three-state) unfolding transition in which the C-terminal repeats unfold at moderate urea concentrations. Removing the C-terminal repeats (Notch ankyrin repeats six and seven) restores equilibrium two-state folding and demonstrates that the high stability of the consensus repeats is propagated into the N-terminal, naturally occurring Notch ankyrin repeats. This stability increase greatly increases the folding rate, and suggests that the transition state ensemble may be repositioned in the chimeric consensus-stabilized proteins in response to local stability.