Structures of designed armadillo-repeat proteins show propagation of inter-repeat interface effects

Designed armadillo-repeat proteins are promising scaffolds for modular peptide-recognition systems. The crystal structures of His-YIIIM4AII, His-YIIIM5AII and YIIIM5AII highlight structural heterogeneity in full-consensus designs and aid the improvement of future constructs.


Introduction
For the design of artificial peptide-binding modules, scaffolds with modular architectures are highly suitable. In particular, the armadillo repeat reveals structural properties that facilitate the design of peptide-binding modules on a rational basis (Andrade et al., 2001;Kajander et al., 2006;Boersma & Plü ckthun, 2011;Reichen, Hansen et al., 2014). In natural armadillo-repeat proteins such as importin-and -catenin, each repeat comprises three -helices that are assembled in a triangular spiral staircase arrangement. All repeats are fused into a single protein with an elongated hydrophobic core (Figs. 1a and 1b). They recognize their target peptides in extended -sheet conformations with very regular binding topologies. The main chain of the peptide is bound in an antiparallel direction by conserved asparagine residues on the concave side of the armadillo-repeat protein (Huber et al., 1997;Conti et al., 1998;Kobe, 1999;Fontes et al., 2003). Differences exist in side-chain preferences because the importin-and -catenin subfamilies recognize peptides with positively and negatively charged side chains, respectively (Conti & Kuriyan, 2000;Ishiyama et al., 2010;Poy et al., 2001).
It is the goal of this protein-engineering project to develop a stable full-consensus armadillo-repeat scaffold. Internal repeats with identical sequences are characteristic of fullconsensus designs. Later, the internal repeats will be functionalized to recognize different amino-acid side chains.
The modularity of the design, which is imposed by the repetitive architecture, should enable us to generate artificial peptide-binding proteins with properties that are precisely tailored according to the length and sequence of the target peptide (Parmeggiani et al., 2008;Reichen, Hansen et al., 2014). Binding proteins with sequence-specific recognition properties for unstructured peptides should be of great interest in research and development because peptide-protein interactions represent 15-40% of all cellular interactions (Petsalaki et al., 2009). Here, many protein-protein interaction scaffolds are unsuitable because they recognize targets based on surface-complementarity properties and thus require a folded counterpart. Conversely, many recognition modules used in intracellular signalling recognize only very short sequences and thus have very low affinity (Pawson & Nash, 2003). Indeed, specific peptide-protein interaction strategies are required to cope with the intrinsic flexibility of unstructured peptides (London et al., 2010).
The first designed armadillo-repeat proteins (dArmRPs) were constructed using a consensus design approach based on 133 and 110 sequences from the importin-and -catenin subfamilies, respectively, in combination with structure-aided modifications of the hydrophobic core (Parmeggiani et al., 2008). They possess the overall composition Y z M n A z , where Y, M and A represent the N-terminal, internal and C-terminal repeats, respectively. The subscripts denote the generation (version) count (z) and the number of internal repeats (n) in roman and arabic numbers, respectively. Since structure-based techniques are vital for this design approach, several structures of proteins from the Y II M n A II and Y III M n A III series have been determined. Initial crystal structures of dArmRPs with second-generation N-and C-caps revealed domain-swapped N-caps, suggesting that the Y II -type N-cap was unstable in solution. To improve the thermodynamic stability of the caps, nine and six mutations were inserted in the N-and C-caps, respectively. These modifications had complementary effects on the thermodynamic stability of the proteins. Introduction of the third-generation N-cap (Y III -type) increased the melting temperature by 4.5 C, but the modifications in the C-cap (A III -type) decreased it by 5.5 C (Madhurantakam et al., 2012). The thermodynamic stabilities of dArmRPs that have so far been designed in this project have been summarized in Reichen, Hansen et al. (2014).
Although the initial crystal structures of His-Y III M 3 A III and His-Y III M 3 A II revealed monomeric proteins (Reichen, Madhurantakam et al., 2014), later studies on Y III M 5 A III (third-generation N-cap and C-cap) without an N-terminal His tag revealed domain-swapped N-caps and C-caps in the presence of calcium ions. However, domain swapping of Y III M 5 A III was not observed either in the absence of calcium ions or in the presence of the His tag because the His tag prevented the unfolding of the N-cap by binding to the neighbouring His-Y III M 5 A III molecule (Reichen, Madhurantakam et al., 2014). To investigate the impact of the cap design on the structural parameters of dArmRPs, particularly in the absence of the His tag, we investigated the crystal structures of the more stable dArmRPs with third-generation N-caps and second-generation C-caps.

Materials and methods
2.1. Cloning, protein expression and purification dArmRPs with cleavable and noncleavable N-terminal His 6 tags have been expressed and purified as described by Reichen, Madhurantakam et al. (2014) with the following modifications: vectors pPank and p148_3C were used for the expression of proteins with and without a cleavable His 6 tag, respectively. The initial designs had noncleavable His 6 tags, but in order to facilitate the elimination of the purification tag, a 3C protease cleavage site was inserted between the His 6 tag and the N-terminus of the N-cap. The amino-acid sequences of the internal and capping repeats are depicted in Fig. 1(c).  The proteins comprise third-generation N-caps, secondgeneration C-caps and four or five internal repeats. All three constructs are full-consensus designs, with internal repeats derived from the M-type internal repeat described in Alfarano et al. (2012). His-Y III M 4 A II and Y III M 5 A II contain M 0 -type internal repeats, whereas His-Y III M 5 A II contains the M 00 -type. In the M 00 -type the aspartic acid at position 1, which was introduced to mimic a potential arginine-binding pocket, was mutated back to the consensus asparagine residue (for all sequences, see Fig. 1c). To improve readability, we refer to M-type internal repeats throughout the text.

Crystallization and structure determination
A Phoenix crystallization robot (Art Robbins Instruments) was used to set up sitting-drop vapour-diffusion experiments in 96-well Corning plates (Corning, New York, USA). Initial crystallization conditions were identified by sparse-matrix screens from Hampton Research (California) and Molecular Dimensions (Suffolk, England), and were later refined by grid screens in which the pH and the precipitant concentrations were varied simultaneously. To confirm the expected peptidebinding site, (KR) 5 peptide was added to Y III M 5 A II in a 1.5:1 molar ratio prior to crystallization. (KR) 5 peptide was used for this experiment because the designed molecular surface of Y III M 5 A II resembled the most conserved importin-peptidebinding site, which recognizes with its core repeats (major and minor binding sites) positive dipeptide motifs composed of lysine and arginine residues. The rationale for this experiment is discussed in Reichen, Hansen et al. (2014). Protein solutions were mixed with reservoir solutions in 1:1, 1:2 or 2:1 ratios (200-300 nl final volume) and the mixtures were equilibrated against 50 ml reservoir solution at 4 C. Reservoir conditions are summarized in Table 1. After washing, the crystals in reservoir solutions supplemented with glycerol were flashcooled in liquid nitrogen.
Data were collected on beamlines X06SA and X06DA at the Swiss Light Source (Paul Scherrer Institute, Villigen, Switzerland) using a Pilatus detector (Dectris, Baden, Switzerland) and a wavelength of 1.0 Å . Diffraction data were processed using MOSFLM (Leslie, 1992) and SCALA (Evans, 2006). Structures were solved by molecular replacement using Phaser (McCoy et al., 2007) together with the following search models. For His-Y III M 4 A II we used the structure of Y III M 3 A II (PDB entry 4db6; Madhurantakam et al., 2012). The refined His-Y III M 4 A II structure was then used to solve the His-Y III M 5 A II and finally the Y III M 5 A II structures. The structures were refined using PHENIX (Adams et al., 2010) and REFMAC5 (Murshudov et al., 2011). For manual model building we used the program Coot (Emsley & Cowtan, 2004). The decrease in R free suggested the use of different refinement strategies for His-Y III M 4 A II and His-Y III M 5 A II . His-Y III M 4 A II was refined without NCS restraints, whereas tight NCS restraints between chains A/B and C/D were applied for the refinement of His-Y III M 5 A II . Figures were prepared using PyMOL (DeLano, 2002). Metal ions were placed manually into strong difference electron-density peaks, taking into account the coordination geometry and the composition of the crystallization buffer. Calcium ions were validated by inspecting the anomalous difference map calculated with phases from the final structure. Water molecules were placed into well defined difference  The crystal structures of His-Y III M 4 A II and His-Y III M 5 A II were refined at 1.8 and 2.0 Å resolution, respectively. In both cases the asymmetric units contain tetramers with 222 point symmetry and very similar topologies. The quaternary structures of His-Y III M 4 A II and His-Y III M 5 A II are governed by calcium ions that connect neighbouring chains in a zipper-like manner and the His 6 tag that binds to the supposed peptidebinding site, albeit in different orientations (see below).
The His-Y III M 4 A II tetramer contains 16 calcium ions. Five calcium ions connect two His-Y III M 4 A II chains in an antiparallel orientation (Fig. 2a). Considering the large size of this interface (average interface area of 1163 Å 2 ) there are relatively few direct hydrogen bonds, and most interactions are made via calcium ions in the loops between helices H2 and H3. The coordination number of each calcium ion in His-Y III M 4 A II is seven, which agrees very well with the statistical analysis of calcium-coordination geometry in protein and small-molecule complexes. Typically, the coordination number of calcium varies between six and eight, with an average length for coordination bonds of between 2.35 and 2.45 Å (Katz et al., 1996). In His-Y III M 4 A II the coordination geometry of calcium differs among ions that are bound to internal or capping repeats.
Ca 2+ ions that bind to internal repeats are contacted by Pro 23 O and Glu 25 OE1 from two symmetry-related chains (superscripts indicate the position in the repeat as indicated in Fig. 1c) and three water molecules (Fig. 2b). Here, Glu 25 contributes one coordination bond (Glu 25 OE1-Ca distance 2.5 Å ). In contrast, calcium ions that bind between an internal repeat and the N-cap are contacted by two water molecules, two O atoms from Glu 25 (Glu 25 OE1-Ca distance of 2.5 Å and Glu 25 OE2-Ca distance of 3.0 Å ), Gln 25 OE1 and Pro 23 O (Fig. 2c). Thus, the replacement of glutamic acid at position 25 by glutamine in the N-cap displaces one water molecule and allows Glu 25 to serve as a bidentate ligand. This observation agrees well with previous data on the statistics of calcium binding, in which it was shown that bidentate binding of carboxylate groups to calcium is particularly prevalent if the coordination number is greater than six (Katz et al., 1996).  (a) The subunits of the His-Y III M 4 A II tetramer are connected via calcium ions. Two chains are sketched as ribbons and coloured as described in Fig. 1(b). Two chains are shown as grey surfaces. Calcium ions are indicated as spheres. Calcium ions binding only to internal repeats are in yellow, those involving the N-cap in light blue and those at the twofold axis in salmon. (b) Calcium-binding site between internal repeats viewed along the axial direction (from the direction of Pro 23# O, which was omitted for clarity). Residues from different chains are shown as sticks with blue and salmon C atoms. Calcium ions and water molecules are depicted as grey and red spheres with reduced atomic radii, respectively. Polar interactions in the pentagonal plane involving the calcium ion are shown as dashed lines in orange. Additional interactions are in yellow. The 2F o À F c and anomalous difference electron-density maps are contoured at 1.3 (light blue) and 4 (green), respectively. (c) Calcium-binding site involving the N-cap. (d) Calcium-binding site at the twofold axis. Colour coding is as described for (b). and five water molecules (Fig. 2d). Furthermore, there are two weakly occupied calcium-binding sites involved in crystal contacts. The His-Y III M 4 A II tetramer is further stabilized by interactions between the N-terminal His 6 tag and the supposed peptide-binding site. This contact is formed by His6, which interacts with Glu 30 and Trp 33 (Glu156 and Trp159) from the third internal repeat, and His8, which interacts with Trp 33 (Trp201) from the fourth internal repeat and Glu 33 (Glu243) from the C-cap (Fig. 3a). Besides the salt bridges between histidine and glutamic acid side chains, the aromatic stacking interaction between His6 and Trp 33 might contribute significant binding energy because the spatial orientation of side chains seen here is frequently found in protein structures (cluster 4 of His-Trp interactions in the atlas of protein sidechain interactions; Singh & Thornton, 1992). Since all four chains of His-Y III M 4 A II are very similar (r.m.s.d. of 0.28 Å for residues 14-246) these interactions are equivalent in all four subunits of the crystallographic tetramer.
In contrast to this, the crystallographic tetramer of His-Y III M 5 A II is less symmetric. Here, chains A/B and C/D are pairwise identical (r.m.s.d. of 0.05 Å ), whereas an r.m.s.d. of 0.85 Å for the comparison between pairs (e.g. chain A with D) suggests substantial differences. Furthermore, His-Y III M 5 A II chains A/B are more similar to His-Y III M 4 A II (r.m.s.d. of 0.72 Å for the superposition of residues 14-210 on the equivalent residues from His-Y III M 4 A II ) than chains C/D (r.m.s.d. of 1.17 Å ). These differences are caused by different contacts within the tetramer. In chains C/D of His-Y III M 5 A II the side chain of Glu198 interacts with His8 from chain D/C (Fig. 3b), whereas in chains A/B the side chain of Glu198 intercalates between internal repeats 3 and 4 and forms a hydrogen bond to the side chain of Gln68 from chains B/A (similar to the interaction shown in Fig. 3a for His-Y III M 4 A II ). As a consequence of this asymmetry, two calcium ions close to the twofold axis, which are present in all four chains of His-Y III M 4 A II (Fig. 2d), are only present in His-Y III M 5 A II chains A/B and are absent from chains C/D.

Structure of Y III M 5 A II without His tag
The structure of Y III M 5 A II without His tag was determined in the absence of calcium ions and refined at 1.95 Å resolution. This structure is most similar to chains C/D of His-Y III M 5 A II (r.m.s.d.s of 1.14 and 0.60 Å for C atoms of residues 14-288 of chains A/B and C/D, respectively). These differences are a consequence of a rigid-body movement of the C-terminal repeats (internal repeats M 4 and M 5 and the C-cap). A superposition of Y III M 5 A II on His-Y III M 5 A II based on the N-cap and internal repeats M 1 -M 3 (residues 14-168) reveals that this part is very similar in all chains. However, in this superposition the C-terminal repeats of  Interface between internal repeats M 3 and M 4 in chain C of His-Y III M 4 A II (a) and His-Y III M 5 A II (b). The dArmRPs are shown in blue and grey and the His 6 tag with salmon C atoms. (c) Superposition based on the N-cap and internal repeats M 1 -M 3 of His-Y III M 5 A II chain A (dark blue), His-Y III M 5 A II chain C (light blue) and Y III M 5 A II (orange). Residues at the M 3 -M 4 interface are labelled. (d) C trace of Y III M 5 A II coloured in green (N-cap), blue (internal repeats) and orange (C-cap). The Leu 32 , Trp 33 and Thr 34 side chains are shown as sticks in blue, grey and green, respectively. Hydrogen bonds and general distances are shown as orange and grey dotted lines, respectively. Distances and conformations of Leu 32 side chains are indicated (tg + , trans/gauche + ; g À t, gauche À /trans). Y III M 5 A II match nicely with the C-terminal repeats of His-Y III M 5 A II chains C/D, but they are shifted towards M 3 in chains A/B (1.4 Å shift of Trp201 CA towards Leu158 CA). This movement can be described as an 8 rotation around an axis that runs parallel to the stacking direction of the C-terminal part and is probably a consequence of different side-chain conformations of Leu158, Trp159, Glu198 and Trp201 at the interface between M 3 and M 4 (Fig. 3c). The structures of His-Y III M 4 A II and Y III M 5 A II represent extreme cases that are most different. In His-Y III M 5 A II these differences are combined into a single structure. His-Y III M 5 A II chains A/B and C/D represent the conformations seen in His-Y III M 4 A II (all chains) and Y III M 5 A II (all chains), respectively. Similar structural plasticity has been observed previously for the comparison of -catenin crystallized in two different crystal forms. For -catenin the C-terminal repeats were rotated 11.5 around an axis that runs approximately parallel to the axis of the superhelix (Huber et al., 1997).
Thus, dArmRPs with second-generation C-caps and thirdgeneration N-caps possess substantial flexibility, particularly for the side chains of Glu 30 , Leu 32 and Trp 33 (equivalent to Glu156, Leu158 and Trp159 in repeat M 3 and Glu198, Leu200 and Trp201 in repeat M 4 ). Experimental structural data for importin-in complex with nuclear localization sequence (NLS) peptides (Conti et al., 1998) and modelling studies on dArmRPs-peptide complexes (Reichen, Hansen et al., 2014) indicate that the superhelix parameters and the conformations of Glu 30 and Trp 33 , which also participate in binding the His 6 tag as outlined above, are important structural features for proper binding of the target peptide. In a first approximation, the curvature of the peptide-binding site can be described by the distances of C atoms at position 33. In the major NLS peptide-binding site of importin-(PDB entry 1bk6; Conti et al., 1998) the distance between C atoms of adjacent Trp 33 residues (e.g. Trp153, Trp195 and Trp237 in repeats 1-3) varies between 8.6 and 8.8 Å . In Y III M 5 A II the average distance between these atoms is 8.82 AE 0.39 Å . However, in Y III M 5 A II the spread between Trp 33 C -atom distances is extremely large, with the largest distance observed between repeats M 3 and M 4 (the distances between Trp159 CA and Trp201 CA are 9.42 Å in chain A and 9.43 Å in chain B). This distance is probably too large for binding the target peptide in the desired conformation and this mismatch is located almost at the centre of the putative peptide-binding site. It is possible that this mismatch is responsible for the fact that the (KR) 5 peptide was not observed in the electron-density map, although it was present during crystallization. Interestingly, the rigid-body movement of the C-terminal part as seen in His-Y III M 4 A II (all chains) and His-Y III M 5 A II (chains C/D) brings this value to the other extreme. Here, the distance of Trp 33 C atoms between repeats M 3 and M 4 is 8.14 AE 0.06 Å , which might be too short for proper binding.
Although Y III M 5 A II is considered to be a full consensus design regarding the sequence of internal repeats, the internal repeats are not identical in terms of structure. These differences can be exerted either by different lattice contacts (Figs. 3a and 3b) or by improper design, which prevents the internal repeats from obtaining a unique conformation throughout the protein. Different distances between adjacent repeats are probably the result of both effects. In particular, the side-chain conformations of buried residues in the hydrophobic core, such as Ile 27 , Leu 32 , Thr 34 , Gly 36 and Ile 38 , mediate the contacts between adjacent repeats. In the structure of Y III M 5 A II the side-chain conformations of Thr 34 , Ile 38 and of course Gly 36 are invariant. The side chain of Thr 34 cross-links internal repeats by forming hydrogen bonds to the main-chain carbonyl groups of Leu 32 and Glu 30 from adjacent repeats. The side chain of Ile 27 adopts mainly gauche À /trans conformations, whereas the side chain of Leu 32 alternates between trans/gauche + and gauche À /trans (Fig. 3d).
This alternation suggests that a uniform conformation of Leu 32 is impossible. In the interface between M 3 and M 4 of Y III M 5 A II , where we observe the largest distance between Trp 33 C atoms, Leu158 CD1 (Leu 32 in M 3 ) and Thr202 OG1 (Thr 34 in M 4 ) are at van der Waals distances (3.86 and 3.97 Å in chains A and B) because the Leu158 side chain adopts a trans/gauche + conformation.
Therefore, steric hindrance between Leu158 and Thr202 might be responsible for increasing the distance between Trp 33 C atoms and for the failure to obtain a dArmRPpeptide complex structure. To adopt a Trp 33 C distance which is similar to the values seen in the major binding site of importin-, Thr202 OG1 would have to move closer to Leu158, but this approach would require a gauche À /trans conformation of the Leu158 side chain. Superposition of Y III M 5 A III (third-generation C-cap; PDB entry 4plq; salmon) on Y III M 5 A II (second-generation C-cap; blue). (a) Residues at the M 3 -M 4 interface. General distances and hydrogen bonds are shown as grey and orange dotted lines, respectively. Distance values refer to Y III M 5 A III . The superposition is based on all C atoms from M 3 . (b) Residues at the M 5 -C-cap interface. Numbers refer to positions in the repeat (Fig. 1c), with subscripts indicating the internal repeat number or the C-cap. Side chains of all residues that differ between Y II and Y III and some residues from the hydrophobic core are shown in stick representation. The superposition is based on all C atoms from M 5 .
Of course, surface-exposed side chains (such as Trp 33 and Glu 30 ) also adopt different rotamers, but it can be assumed that these differences affect inter-repeat distances to a minor extent because the environments of surface-exposed side chains are usually less densely packed than the environments of buried side chains. However, some side-chain conformations of buried and surface-exposed residues are coupled. For example, the conformation of Trp 33 is linked to the conformation of Leu 32 in the preceding repeat. In repeats M 1 and M 3 Leu 32 adopts trans/gauche + conformations and Trp 33 in repeats M 2 and M 4 is trans/+90 , whereas in repeats M 2 and M 4 Leu 32 is gauche À /trans and Trp 33 adopts trans/À105 conformations in repeats M 3 and M 5 (Fig. 3d). Only Trp243 in chain B deviates from this general observation.

Comparison of dArmRPs with second-generation and third-generation C-caps
The crystal structures of Y III M 5 A III with and without a His 6 tag and third-generation C-caps have been published recently (Reichen, Madhurantakam et al., 2014). Y III M 5 A III without a His 6 tag but crystallized in the presence of calcium revealed domain-swapped N-and C-caps. Since Y III M 5 A II without a His 6 tag and a second-generation C-cap did not crystallize in the presence of calcium, it remains unclear whether the redesign of the C-cap was responsible for calcium-induced domain swapping.
Interestingly, Y III M 5 A III also shows an extended distance between Trp 33 C atoms of internal repeats M 3 and M 4 (distance between Trp159 CA and Trp201 CA of 8.86 Å ), a short distance between Thr202 OG1 and Leu158 CD2 of 3.91 Å and no electron density for the (KR) 5 peptide, although it was present during crystallization (Reichen, Madhurantakam et al., 2014). On the other hand, Leu158 shows the gauche À /trans side-chain conformation, which is trans/gauche + in Y III M 5 A II , probably because Glu198 forms an additional hydrogen bond to Gln155 O (Fig. 4a).
For dArmRPs with three internal repeats it was shown that the redesign of the C-cap (from A II to A III ) decreases the melting temperature by 5.5 C (Madhurantakam et al., 2012), and a domain-swapped C-cap was observed for Y III M 5 A III (Reichen, Madhurantakam et al., 2014). Both observations suggest that Y III M 5 A III is less stable than Y III M 5 A II . A superposition of Y III M 5 A III (PDB entry 4plq) and Y III M 5 A II based on the last internal repeat suggests that this destabilization might be owing to subtle rearrangements in the hydrophobic core between internal repeats M 4 and M 5 and the C-cap. Three out of six mutations that were introduced at the C-cap are solvent-exposed and do not seem to have a significant effect on the structure. However, Lys 15 !Ala, His 22 !Ser and Leu 38 !Ile mutations cause a gentle rearrangement of the C-cap (Fig. 4b). This rearrangement has implications for the packing of side chains in the hydrophobic core. In the more stable Y III M 5 A II structure the side chains of Leu 16 , Leu 20 and Val 7 adopt a uniform distribution of side-chain rotamers in all repeats. Val 7 adopts a trans conformation. Leu 16 and Leu 20 are always gauche À /trans. In Y III M 5 A III this crystal-like arrange-ment is perturbed by the C-cap. In Y III M 5 A III the side chains of Leu 16 , Leu 20 and Val 7 adopt the same conformations as in Y III M 5 A II only in the N-terminal part, whereas in the C-terminal part their conformations are clearly different. For Leu 32 the situation is inverted. In Y III M 5 A III the rotamer distribution of Leu 32 is uniform, whereas in Y III M 5 A II alternating Leu 32 conformations are observed (Fig. 3d). Uniform distributions of rotamers are frequently observed in polypeptides with very high thermodynamic stabilities, such as amyloid fibrils (Nelson et al., 2005) and -helix proteins (Schulz & Ficner, 2011). Therefore, it can be assumed that the uniform distribution of side-chain rotamers is related to the stability of dArmRPs and vice versa. On the other hand, the deterioration of uniformity, as caused by the third-generation C-cap, is linked to destabilization of the protein.
In conclusion, this detailed investigation of the different versions of dArmRPs has shown that small differences in packing between repeats, notably between internal repeats and the caps, can make the protein susceptible to perturbations caused by crystal contacts and ions used in crystallization, indicating a lack of rigidity. This leads to a surprising long-range effect of changes in the C-cap and helps to explain the astonishing observation that a full-consensus design does not necessarily generate a unique repeat conformation. Although the internal repeats are chemically absolutely identical, their conformations lack uniformity. The current analysis suggests that future improvements of an armadillorepeat-based peptide-recognition system will have to take three considerations into account. (i) In particular, the deletion of the His tag seems to be crucial for liberating the presumed peptide-binding site. (ii) The second-generation C-cap presented here seems to be superior to the thirdgeneration C-cap, which was initially believed to be more advanced. (iii) The choice of amino acids at the inter-repeat interface, particularly at positions 27, 32 and 34, should be reconsidered because the side chains at these positions show substantial conformational heterogeneity.