In this chapter we review recent studies of repeat proteins, a class of proteins consisting of tandem arrays of small structural motifs that stack approximately linearly to produce elongated structures. We discuss the observation that, despite lacking the long-range tertiary interactions that are thought to be the hallmark of globular protein stability, repeat proteins can be as stable and as coorperatively folded as their globular counterparts. The symmetry inherent in the structures of repeat arrays, however, means there can be many partly folded species (whether it be intermediates or transition states) that have similar stabilities. Consequently they do have distinct folding properties compared with globular proteins and these are manifest in their behaviour both at equilibrium and under kinetic conditions. Thus, when studying repeat proteins one appears to be probing a moving target: a relatively small perturbation, by mutation for example, can result in a shift to a different intermediate or transition state. The growing literature on these proteins illustrates how their modular architecture can be adapted to a remarkable array of biological and physical roles, both in vivo and in vitro. Further, their simple architecture makes them uniquely amenable to redesign—of their stability, folding and function—promising exciting possibilities for future research.

Introduction

Repeat proteins, such as ankyrin repeats, leucine rich repeats (LRR) and tetratricopeptide repeats (TPR), consist of tandem arrays of a small structural motif of ∼20-40 amino acids that stack in a roughly linear fashion, creating elongated and superhelical architectures. They are mostly found in arrays of four, up to tens of repeats and they can comprise an entire protein or a domain within a larger protein. Repeat protein structures have simple topologies composed entirely of short-range interactions between residues within a repeat or between adjacent repeats, in striking contrast to the more complex topologies of globular proteins that are stabilized by contacts between residues distant in sequence. They are ubiquitous and highly versatile, mediating molecular recognition in many different biological processes. A picture is emerging, from work carried out in the last five years, of the simplicity of this modular architecture which has enabled the following: (1) proteins containing consensus repeat motifs have been produced with enhanced thermodynamic stabilities compared with their natural counterparts; (2) designed consensus repeat proteins have been used successfully as scaffolds for engineering novel binding specificities; (3) repeat proteins have folding pathways that are amenable to design. None of these features have been found with any consistency for globular proteins. By far the most work has been carried out on ankyrin repeat proteins, followed by TPR proteins and these two will therefore be the focus of this review.

Repeat Protein Structures

As with globular proteins, the repeat protein family can be broken down further into sub-families based on their secondary structure content. A range of different repeat proteins are shown in Figure 1. The most common forms of α-helical repeat proteins are ankyrin repeats and TPR repeats. Ankyrin repeats (named after the protein Ankyrin in which these repeats were first identified¹) have an L-shaped profile, with a long loop/β-hairpin sitting at right angles to a pair of antiparallel α-helices (Fig. 1a and b). The helices of one repeat pack against the helices of an adjacent repeat, with the interface between repeats consisting of mainly hydrophobic interactions stabilising the helices and hydrogen bonding stabilising the long loop region. The two helices of the protein are of different lengths causing curvature in the structure with each repeat rotated 2-3° counterclockwise relative to the previous one. The tetratricopeptide repeat (TPR) consists of a 34 residue motif ^2,3 that adopts an antiparallel helix-turn-helix arrangement without any long connecting loops unlike ankyrin repeats. The packing of a pattern of small and large hydrophobic residues results in a rather splayed arrangement of the α-helices. The crystal structure of protein phosphatase 5 contains three TPR repeats and forms a right-handed superhelical structure with a continuous helical groove⁴ (Fig. 1c). The groove is the proposed site of protein-protein interactions. The curvature that results from arrays of large numbers of repeats is particularly well-illustrated by the structure of the procine ribonuclease inhibitor⁵(Fig. 1g).

Figure 1

Repeat protein architectures. Interaction partners are colored in grey. (a) ANK: the ternary complex between p18^INK4c, Cdk6 and K-cyclin (1G3N). (b) ANK: The transcription factor complex between NFκB and IκBα (1IKN) (c) TPR: The (more...)

Interestingly, not all repeat proteins form linear elongated structures. The prokaryotic TPR protein N1p1 forms a globular structure in which repeats from the N-terminus forming a central hydrophobic core and the C-terminal repeats forming the outer solvent exposed surface, with the entire protein resembling a spiral.⁶ Further, WD40 repeats form propeller-like structures with each repeat folding into a β-sheet resembling a blade of the propeller⁷ (Fig. 1h).

Repeat Proteins as Mediators in Molecular Recognition

The large and regular surfaces of repeat proteins lend them ideally to mediating molecular recognition events. Repeat proteins provide a central scaffold with a conserved sequence motif and typically have highly variable sequences in loops presented on the surface. In this way, the loops can provide varied interaction surfaces in terms of geometry as well as functional group content. Repeat proteins are frequently found in proteins containing other domains and they may serve to recruit substrates in these cases. An important class of such proteins are the F-box proteins.⁸ SCF complexes are an important class of E3 ubiquitin ligases that target key cell cycle regulators and transcription factors to the proteasome for degradation. SCF complexes consist of two invariable, core subunits and a third, variable, subunit, the F-box protein that recruits substrates for ubiquitination by an associated E2 enzyme. F-box proteins have an F-box domain that binds to the core SCF subunits and a substrate recognition domain, in many cases composed of LRRs or WD40 repeats.

Ankyrin repeats have been extensively studied in terms of their protein-protein interactions. For example, the INK4 family of ankyrin repeat proteins specifically inhibit the cyclin-dependent kinases (CDK) of the cell cycle (Fig. 1a). There are four members of the INK4 family of CDK inhibitors: p15INK4B, p16INK4A, p18INK4C, p19INK4D. All four proteins are biochemically indistinguishable with respect to inhibition of CDK4/6. Understanding the mechanism of molecular recognition of these proteins is made more important by the fact that a common event in tumorigenesis is mutation of the regulatory proteins involved in the G1-S transition. The INK4 family of proteins specifically inhibit the cyclin-dependent kinases CDK4 and CDK6 responsible for initiating this transition. Insertions in the ankyrin repeat motif are common and usually found in the loop regions. In the case of the INK4 proteins, the first helix in the second repeat is shorter than the consensus one.

The ankyrin repeats of the protein IκBα are an interesting example of the phenomenon of protein folding induced by binding, as is seen in an increasing number of protein-protein interactions. IκBα is an inhibitor of the transcription factor NF-κB and the NF-κB-binding domain of IκBα has six ankyrin repeats (Fig. 1b). The structure of IκBα is only known in complex with NF-κB but extensive biophysical analysis revealed that although the protein is compactly folded and has all of its secondary structural elements it nevertheless has some highly dynamic, molten-globule-like regions in both free and complexed forms.⁹ Subsequent analysis using hydrogen/deuterium exchange monitored by mass spectrometry showed that the β-hairpins of ankyrin repeats 5 and 6 only fold upon binding to NF-κB.^10,11 The authors propose that this feature of IκBα may be important for several aspects of its function that are critical for the tight regulation of NF-κB activity. In particular, the flexibility of the hairpins may help it to bind to different NF-κB dimers resulting in the transcription of different genes and also facilitate the dissociation of NF-κB from DNA.

Another example of the molecular recognition capabilities of repeat proteins is found in nuclear transport (reviewed in 12,13). The import of cargos into the nucleus of cells is dependent on Armadillo repeat- and HEAT repeat-containing proteins known as the importins. The Armadillo repeat was first identified in the Drosophila melanogaster gene product Armadillo.¹⁴ These proteins consist of tandem repeats of an approximately 40-residue motif which folds into a three-helix bundle. The approximately 40-residue HEAT motif consists of only two helices. The HEAT motif was initially found in a diverse range of proteins, including the four from which its name derives: huntingtin, elongation factor 3, the PR65/A subunit of protein phosphatase 2A and the lipid kinase Tor (target of rapamycin).¹⁵ In both armadillo- and HEAT-repeat proteins, the motifs stack together to form an elongated super-helix. Cargoes destined for the nucleus and displaying the nuclear localisation signal (NLS),^16-19 a short predominantly positively charged peptide, bind importin-α, a protein containing 10 armadillo repeats in which the super-helix creates a narrow internal groove that envelops the NLS and displays its own positively charged peptide²⁰ (Fig. 1e). This importin-α peptide interacts with a 19-HEAT repeat protein, importin-β/β-karyopherin²¹ (Fig. 1d). The resulting large complex enables the cargo to translocate through the nuclear pore. Not only are these interactions extremely specific for the NLS peptide, but the outer surface of these proteins also allow the complex to dissolve into the central channel of the pore itself in a highly selective fashion. A few cargo proteins bind directly to importin-β rather than via importin-α. Once in the nucleus, RanGTP binds to importin-β thereby dissociating the importin-αβ complex and leading to release of the cargo. Another 19-HEAT repeat protein, CAS, then exports importin-α out of the nucleus so that it can be used again. Crystal structures reveal that the HEAT repeats of both importin-β and CAS function as tightly wound springs with the curvature created by the packing of the individual α-helices and thereby the twist of the super-helix, changing dramatically depending on whether they are in the free or complexed form. It is thought that this flexibility enables them to bind to a range of different partners by wrapping around them with varying helicoidal pitch.

The regular presentation of surface residues provided by the repeat protein architecture lends itself to roles other than just mediating interactions between proteins. For example, the antifreeze proteins of some invertebrates form fascinating structures that are able to directly bind to ice crystals. These antifreeze proteins, found in invertebrates such as the Tenebrio molitor beetle, are composed of tandem 12-residue repeats (TCTxSxxCxxAx) which form linear beta-helical structures which are either square or triangular in cross section²² (Fig. 1f). Remarkably, these beta-helical structures present a regular array of optimally orientated binding pockets which bind water molecules at the edges of ice crystals, thereby preventing further ice crystal growth and therefore damage to the organism.

Designing Repeat Proteins

A number of groups have taken advantage of the symmetry in linearly repeating structures to successfully design novel proteins that have been found to have increased stability relative to the natural counterparts. To date, they have all applied, at least in part, the concept of consensus design²³—engineering a protein to have the most common residue as each position as determined by multiple sequence alignment. The rationale is that the residues that are most highly conserved among proteins of a particular fold will be those that are important for the stability of that fold, although there are a number of reasons why this rationale may be imperfect.²⁴ Two groups have designed consensus ankyrin repeat proteins, making use of the relatively high sequence homology within ankyrin repeats. Pluckthun's group used libraries assembled from N- and C-terminal capping repeats and an internal repeat consensus sequence with 27 fixed positions and the remaining 6 randomized. Six of the library members were chosen at random, containing between four and six repeats.^25-27 Crystallography of one of the proteins showed that it adopted the correct fold.^28,29 The proteins were more stable (as measured by equilibrium chemical denaturation experiments) than natural ankyrin repeat proteins of similar sizes and five of the six were monomeric. The most stable protein had six repeats and a free energy of unfolding that was greater than 20 kcal.mol⁻¹.²⁵ A comparison of the hydrophobic core packing of the designed ankyrin repeat structure with natural ankyrin repeats did not reveal any significant differences. The authors suggest that elimination of insertions, commonly found in the loop regions and used for molecular recognition, may contribute to the enhanced stability of these designed proteins relative to their natural counterparts; the loop regions form a greatly improved, regular network of hydrogen bonds. Increased stability is also likely to come from the conserved TPLH motif in the first helix of the ankyrin repeat which again results in a network of hydrogen bonds extending throughout the molecule, as well as from the tight packing of hydrophobic residues between the α-helices. A similar approach was used to design leucine-rich repeat protein libraries. As for the consensus ankyrin repeat proteins, the leucine-rich repeat proteins were found to be expressed at very high levels in E. coli, soluble, monomeric and stable.³⁰ Peng's group designed a consensus ankyrin repeat sequence that differed in four positions from Pluckthun's³¹. The other major difference was that they did not use capping repeats that were different from the internal repeats. They built proteins consisting of 1-4 ankyrin repeats and X-ray crystal structures of the two larger proteins showed that they adopted the correct fold.³¹ The designed proteins were also found to have high thermal stability but they were only soluble in acidic conditions. The solubility at neutral pH was improved by making substitutions of surface leucine residues for arginines.

Pluckthun's group have since gone on to show that the idea of using consensus ankyrin repeats as scaffolds on which to graft novel binding specificities can have great success. The residues to be used for molecular recognition are located on the β-turn and first helix of each repeat, creating a large and modular surface. Noteworthy are designed 4-ankyrin repeat proteins (DARPins) selected against human epidermal growth factor receptor 2 with high specificity and low nanomolar affinities.³² The interaction was subsequently improved to a 90 nM affinity using error-prone PCR and stringent off-rate selection.³³

Regan's group have made novel TPR proteins by consensus design. They constructed proteins containing 1.5-3.5 TPR motifs³⁴ and additionally incorporated an N-capping, helix-stabilising GNS sequence at the N-terminus and an extra solvating helix at the C-terminus. The rationale for the latter addition was that similar helices were present in all of the structures of TPR-containing proteins and were found to increase solubility. The 2.5- and 3.5-TPR repeat proteins folded into the correct structures and had high thermal stabilities.³⁵ Whereas the enhanced stability of the consensus-designed ankyrin repeat proteins appears to come from the regularised hydrogen bonding networks, the stabilising interactions in the consensus TPR structures are predominantly the large hydrophobic residues that force the α-helices apart into the characteristic elongated architecture. This group then took the consensus TPR repeats and redesigned them to bind to the C-terminal peptide of the molecular chaperone Hsp90 (a natural TPR binding partner).³⁶ The designed protein had greater specificity than the natural TPR proteins.

Several studies have recently shown that the stabilities of naturally occurring ankyrin repeat proteins can be enhanced by using the consensus sequence.^37-39 For example, for the 7-ankyrin repeat domain of Notch, replacement of the two C-terminal repeats by consensus repeats increased the stability of the protein by almost 6 kcal.mol⁻¹.³⁸ Substitution by consensus residues of two residues in the N-terminal repeat of the 4-ankyrin repeat protein myotrophin increased the stability by over 2 kcal.mol⁻¹.³⁹ The 6-ankyrin repeat region of IkBa has only marginal stability; engineering of three residues, one in each of three of the repeats, to conform to the extended motif GXTPLHLA of the consensus (that creates a hydrogen bonding network as described above) resulted in an increase in stability of 4.5 kcal mol⁻¹.³⁷

Biophysical Properties of Repeat Proteins

The distinctive architecture of repeat proteins compared with globular proteins makes them a particular interesting target for biophysical analysis. The simple, modular nature of the structures and the lack of long-range interactions (which are thought to be contribute to the cooperative folding of globular proteins), pose a number of questions. First, are repeat proteins more or less stable than globular proteins of similar size? Second, are repeat structures less cooperatively folded given the lack of long-range contacts? Third, are the differences in the structures of repeat proteins compared with globular proteins reflected in their folding mechanisms? In particular, it has been shown that there is a correlation between folding rate and proportion of short-range contacts in the native structure because there is a smaller entropic cost of closing a short loop to form an interaction between residues close in sequence than closing a long loop between sequence-distant residues;⁴⁰ so, do repeat proteins fold faster than globular ones because of the predominance of short-range contacts? And, are there multiple folding pathways accessible due to the linear symmetry of their structures?

Summary

Repeat proteins have turned out to be a fascinating class of structures, with particular appeal to those interested in protein folding, engineering and design. They are modular and therefore much more amenable to redesign than are globular proteins and yet this feature does not appear to compromise cooperativity, a hallmark of globular proteins that is thought to be important in making them sufficiently robust to maintain them in their native structures over their functional lifetimes. The recent studies reviewed here reveal design-ability in every aspect of repeat proteins, including their stability, folding and function. It will be interesting to see whether these striking properties, observed to date for the relatively small ankyrin and TPR repeats, are recapitulated in the behaviour of larger repeat motifs such as Armadillo and HEAT. We also look forward with anticipation to seeing how these properties will be exploited and manipulated in the future.

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