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.     

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.     

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.     

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.     

Structure and Notch receptor binding of the tandem WWE domain of Deltex

Deltex is a cytosolic effector of Notch signaling thought to bind through its N-terminal domain to the Notch receptor. Here we report the structure of the Drosophila Deltex N-terminal domain, which contains two tandem WWE sequence repeats. The WWE repeats, which adopt a novel fold, are related by an approximate two-fold axis of rotation. Although the WWE repeats are structurally distinct, they interact extensively and form a deep cleft at their junction that appears well suited for ligand binding. The two repeats are thermodynamically coupled; this coupling is mediated in part by a conserved segment that is immediately C-terminal to the second WWE domain. We demonstrate that although the Deltex WWE tandem is monomeric in solution, it forms a heterodimer with the ankyrin domain of the Notch receptor. These results provide structural and functional insight into how Deltex modulates Notch signaling, and how WWE modules recognize targets for ubiquitination.     

The tolerance of a modular protein to duplication and deletion of internal repeats

Ankyrin repeat polypeptides contain repeated structural elements that pack to produce modular architectures lacking in close contacts between distant segments of the polypeptide chain. Despite this lack of sequence-distant contacts, ankyrin repeat polypeptides have been shown to fold in a cooperative manner. To determine the distance over which cooperative interactions can be propagated in a repeat protein, and to investigate the tolerance to internal duplication and deletion of modules, we have constructed a series of ankyrin repeat variants of the Notch ankyrin domain in which repeat number is varied by duplication and deletion of internal repeats. A construct with two copies of the fifth ankyrin repeat shows a modest increase in stability compared to the parent construct and retains apparent two-state unfolding behavior. Although constructs containing three and four copies of the fifth repeat retain this increased resistance to urea, they exhibit broad, multi-state unfolding transitions compared to the parent construct. For the Notch ankyrin domain, these larger constructs may represent a limit beyond which full cooperativity cannot be maintained. Deletions of internal repeats from the Notch ankyrin domain significantly destabilize the domain. This severe destabilization, which is larger than that resulting from end-repeat deletion, may arise from unfavorable interactions within the new non-native interfaces produced by internal repeat deletion. These results demonstrate both an asymmetry between the duplication and deletion of internal repeats, and a difference between deletion of internal and end-repeats, suggesting preferred mechanisms for evolution of repeat proteins.     

Evolutionary analysis of vertebrate Notch genes

We have conducted an evolutionary analysis of Notch genes of the vertebrates Danio rerio and Mus musculus to examine the expansion and diversification of the Notch family during vertebrate evolution. The existence of multiple Notch genes in vertebrate genomes suggests that the increase in Notch signaling pathways may be necessary for the additional complexity observed in the vertebrate body plan. However, orthology relationships within the vertebrate Notch family indicate that biological functions are not fixed within orthologous groups. Phylogenetic reconstruction of the vertebrate Notch family suggests that the zebrafish notch1a and 1b genes resulted from a duplication occurring around the time of the teleost/mammalian divergence. There is also evidence that the mouse Notch4 gene is the result of a rapid divergence from a Notch3-like gene. Investigation of the ankyrin repeat region sequences showed there to be little evidence for gene conversion events between repeat units. However, relationships between repeats 2-5 suggest that these repeats are the result of a tandem duplication of a dual repeat unit. Selective pressure on maintenance of ankyrin repeat sequences indicated by relationships between the repeats suggests that specific repeats are responsible for particular biological activities, a finding consistent with mutational studies of the Caenorhabditis elegans gene glp-1. Sequence similarities between the ankyrin repeats and the region immediately C-terminal of the repeats further suggests that this region may be involved in the modulation of ankyrin repeat function.     

Structural Determinants for Improved Stability of Designed Ankyrin Repeat Proteins with a Redesigned C-Capping Module

Designed ankyrin repeat proteins (DARPins) that specifically bind to almost any target can be obtained by ribosome display or phage display from combinatorial libraries. Although DARPins are already very stable molecules, molecular dynamics simulations, equilibrium denaturation experiments, structural studies, and recent NMR experiments suggested that the unfolding of the original C-terminal capping repeat (C-cap), taken from a natural ankyrin repeat protein, limits the stability of the initial DARPin design. Several point mutations had been introduced to optimize the C-cap and were shown to indeed further increase the stability of DARPins. We now determined crystal structures of DARPins with one or three full-consensus internal repeats (NI₁C or NI₃C) between an N-terminal capping repeat and mutants of the C-cap. An NI₁C mutant, in which the C-cap was only extended by three additional helix-forming residues, showed no structural change but reduced B-factors in the C-cap. An NI₃C C-cap mutant carrying five additional mutations in the interface to the preceding repeat, previously designed by using the consensus sequence as a guide, showed a rigid-body movement of the C-cap towards the internal repeat. This movement results in an increased buried surface area and a superior surface complementarity and explains the improved stability in equilibrium unfolding, compared to the original C-cap. A C-cap mutant with three additional mutations introducing suitably spaced charged residues did not show formation of salt bridges, explaining why its stability was not increased further. These structural studies underline the importance of repeat coupling for stability and help in the further design of this protein family.     

Crystal structure of the human TRPV2 channel ankyrin repeat domain

TRPV channels are important polymodal integrators of noxious stimuli mediating thermosensation and nociception. An ankyrin repeat domain (ARD), which is a common protein-protein recognition domain, is conserved in the N-terminal intracellular domain of all TRPV channels and predicted to contain three to four ankyrin repeats. Here we report the first structure from the TRPV channel subfamily, a 1.7 A resolution crystal structure of the human TRPV2 ARD. Our crystal structure reveals a six ankyrin repeat stack with multiple insertions in each repeat generating several unique features compared with a canonical ARD. The surface typically used for ligand recognition, the ankyrin groove, contains extended loops with an exposed hydrophobic patch and a prominent kink resulting from a large rotational shift of the last two repeats. The TRPV2 ARD provides the first structural insight into a domain that coordinates nociceptive sensory transduction and is likely to be a prototype for other TRPV channel ARDs.     

Characterization and further stabilization of designed ankyrin repeat proteins by combining molecular dynamics simulations and experiments

Multiple molecular dynamics simulations with explicit solvent at room temperature and at 400 K were carried out to characterize designed ankyrin repeat (AR) proteins with full-consensus repeats. Using proteins with one to five repeats, the stability of the native structure was found to increase with the number of repeats. The C-terminal capping repeat, originating from the natural guanine-adenine-binding protein, was observed to denature first in almost all high-temperature simulations. Notably, a stable intermediate is found in experimental equilibrium unfolding studies of one of the simulated consensus proteins. On the basis of simulation results, this intermediate is interpreted to represent a conformation with a denatured C-terminal repeat. To validate this interpretation, constructs without C-terminal capping repeat were prepared and did not show this intermediate in equilibrium unfolding experiments. Conversely, the capping repeats were found to be essential for efficient folding in the cell and for avoiding aggregation, presumably because of their highly charged surface. To design a capping repeat conferring similar solubility properties yet even higher stability, eight point mutations adapting the C-cap to the consensus AR and adding a three-residue extension at the C-terminus were predicted in silico and validated experimentally. The in vitro full-consensus proteins were also compared with a previously published designed AR protein, E3_5, whose internal repeats show 80% identity in primary sequence. A detailed analysis of the simulations suggests that networks of salt bridges between β-hairpins, as well as additional interrepeat hydrogen bonds, contribute to the extraordinary stability of the full consensus.     

Stabilizing ionic interactions in a full-consensus ankyrin repeat protein

Full-consensus designed ankyrin repeat proteins (DARPins), in which randomized positions of the previously described DARPin library have been fixed, are characterized. They show exceptionally high thermodynamic stabilities, even when compared to members of consensus DARPin libraries and even more so when compared to naturally occurring ankyrin repeat proteins. We determined the crystal structure of a full-consensus DARPin, containing an N-capping repeat, three identical internal repeats and a C-capping repeat at 2.05 Å resolution, and compared its structure with that of the related DARPin library members E3_5 and E3_19. This structural comparison suggests that primarily salt bridges on the surface, which arrange in a network with almost crystal-like regularity, increase thermostability in the full-consensus NI₃C DARPin to make it resistant to boiling. In the crystal structure, three sulfate ions complement this network. Thermal denaturation experiments in guanidine hydrochloride directly indicate a contribution of sulfate binding to the stability, providing further evidence for the stabilizing effect of surface-exposed electrostatic interactions and regular charge networks. The charged residues at the place of randomized residues in the DARPin libraries were selected based on sequence statistics and suggested that the charge interaction network is a hidden design feature of this protein family. Ankyrins can therefore use design principles from proteins of thermophilic organisms and reach at least similar stabilities.     

SEL-10 is an inhibitor of notch signaling that targets notch for ubiquitin-mediated protein degradation

Notch receptors and their ligands play important roles in both normal animal development and pathogenesis. We show here that the F-box/WD40 repeat protein SEL-10 negatively regulates Notch receptor activity by targeting the intracellular domain of Notch receptors for ubiquitin-mediated protein degradation. Blocking of endogenous SEL-10 activity was done by expression of a dominant-negative form containing only the WD40 repeats. In the case of Notch1, this block leads to an increase in Notch signaling stimulated by either an activated form of the Notch1 receptor or Jagged1-induced signaling through Notch1. Expression of dominant-negative SEL-10 leads to stabilization of the intracellular domain of Notch1. The Notch4 intracellular domain bound to SEL-10, but its activity was not increased as a result of dominant-negative SEL-10 expression. SEL-10 bound Notch4 via the WD40 repeats and bound preferentially to a phosphorylated form of Notch4 in cells. We mapped the region of Notch4 essential for SEL-10 binding to the C-terminal region downstream of the ankyrin repeats. When this C-terminal fragment of Notch4 was expressed in cells, it was highly labile but could be stabilized by the expression of dominant-negative SEL-10. Ubiquitination of Notch1 and Notch4 intracellular domains in vitro was dependent on SEL-10. Although SEL-10 interacts with the intracellular domains of both Notch1 and Notch4, these proteins respond differently to interference with SEL-10 function. Thus, SEL-10 functions to promote the ubiquitination of Notch proteins; however, the fates of these proteins may differ.     

The crystal structure of gankyrin, an oncoprotein found in complexes with cyclin-dependent kinase 4, a 19 S proteasomal ATPase regulator, and the tumor suppressors Rb and p53

Gankyrin is a 25-kDa hepatocellular carcinoma-associated protein that mediates protein-protein interactions in cell cycle control and protein degradation. It has been reported to form complexes with cyclin-dependent kinase 4, retinoblastoma protein, the S6b ATPase subunit of the 19 S regulator of the 26 S proteasome, and Mdm2, an E3 ubiquitin ligase involved in p53 degradation. It is the first protein described to bind both to the 26 S proteasome and to proteins in other complexes containing cyclin-dependent kinase(s) and p53 ubiquitylating activities, thus providing a mechanism for delivering cell cycle regulating machinery and ubiquitylated substrates to the proteasome for degradation. Gankyrin contains a 33-residue motif known as the ankyrin repeat that occurs five and a half to six times in the sequence. As a step toward understanding gankyrin interactions with its protein partners we have determined its three-dimensional crystal structure to 2.0-A resolution. It reveals that the entire 226-residue gankyrin polypeptide folds into seven ankyrin repeat elements. The ankyrin repeats, consisting of an antiparallel beta-hairpin followed by a perpendicularly oriented helix-loop-helix, pack side-by-side, creating an extended curved structure with a groove running across the long concave surface. Comparison with the structures of other ankyrin repeat proteins suggests that interactions with partner proteins are mediated by residues situated on this concave surface.