Báo cáo khoa học: Crystal structure of a designed tetratricopeptide repeat module in complex with its peptide ligand pot

Crystal structure of a designed tetratricopeptide repeat module in complex with its peptide ligand Aitziber L. Cortajarena 1 , Jimin Wang 1 and Lynne Regan 1,2 1 Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT, USA 2 Department of Chemistry, Yale University, New Haven, CT, USA Introduction The basic tetratricopeptide (TPR) repeat comprises 34 amino acids that adopt a helix–turn–helix structure [1,2]. We refer to the two tandem helices as the A-helix and B-helix. In tandem arrays of TPR repeats, the helices stack to form superhelical structures that dis- play two surfaces: a concave binding face, and a con- vex back face. The natural role of TPR proteins is to mediate protein–protein interactions. Modules with three tandem TPR repeats are by far the most com- mon in nature, and presumably represent the minimal functional binding unit [1]. The simple modular nature of TPR proteins makes them ideal scaffolds for protein design studies. We designed a TPR protein, named CTPR3, com- posed of three repeats of a consensus TPR sequence, and solved its crystal structure at 1.6 A ˚ resolution [3]. The structural alignment of CTPR3 with natural 3-TPR domains clearly shows that its overall structure is almost identical, with backbone rmsd values between 1.1 A ˚ and 1.6 A ˚ for the pairwise alignments [4]. CTPR3 is significantly more stable than natural TPR domains [3], and this enabled us to introduce muta- tions onto this framework without compromising its thermodynamic stability. Starting with CTPR3 as a structural scaffold, we created a protein (CTPR390) that incorporates heat shock protein (Hsp)90-binding residues, grafted from natural Hsp90-binding TPR domains, onto the con- cave ligand-binding face of the domain (A-helices) [5]. We showed that CTPR390 binds to the C-terminal peptide of Hsp90 [5] specifically, with moderate affinity (K d of 200 lm). We also demonstrated that the binding affinity could be modulated, and enhanced, by fine- tuning the long-range electrostatic interactions through modifying the charge on the back face of the protein [6]. Finally, by introducing the designed domain into Keywords crystal structure; Hsp90; protein design; repeat proteins; tetratricopeptide repeat (TPR) Correspondence L. Regan, Department of Molecular Biophysics & Biochemistry, Yale University, New Haven, CT 06520, USA Fax: +1 203 432 5175 Tel: +1 203 432 9843 E-mail: lynne.regan@yale.edu Website: http://www.yale.edu/reganlab/ (Received 26 October 2009, revised 25 November 2009, accepted 16 December 2009) doi:10.1111/j.1742-4658.2009.07549.x Tetratricopeptide repeats (TPRs) are protein domains that mediate key protein–protein interactions in cells. Several TPR domains bind the C-ter- mini of the chaperones heat shock protein (Hsp)90 and ⁄ or Hsp70, and exchange of such binding partners is key for the heat shock response. We have previously described the design of a TPR protein that binds tightly and specifically to the C-terminus of Hsp90, and in doing so, is able to inhibit chaperone function in vivo. Here we present the X-ray crystal struc- ture of the designed TPR domain (CTPR390) in complex with its peptide ligand – the C-terminal residues of Hsp90 (peptide MEEVD). This struc- ture reveals two interesting aspects of the TPR modules. First, a new pack- ing arrangement of 3-TPR modules is observed. The TPR units stack against each other in an unusual fashion to form infinite superhelices in the crystal. Second, the structure provides insights into the molecular basis of TPR–ligand recognition. Abbreviations ASU, asymmetric unit; Hsp, heat shock protein; TPR, tetratricopeptide repeat. 1058 FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS mammalian cells, we showed that it inhibited Hsp90 function, presumably by preventing Hsp90 from form- ing a complex with the TPR2A domain of Hsp- organizing protein (HOP) [6]. Here we describe in detail the X-ray crystal structure of the designed Hsp90-binding TPR module, CTPR390, in complex with the peptide MEEVD, which corre- sponds to the five C-terminal residues of Hsp90. We discuss the unusual superhelical head-to-tail crystal packing between CTPR390 molecules, and compare it with the superhelical packing observed for longer TPR arrays (CTPR8 and CTPR20) [7]. Finally, we analyze the TPR–peptide interaction in detail, thus providing a structural comparison of natural and designed peptide recognition by TPR modules. This work provides key insights into the ‘functional grafting’ design strategy, and also sets the stage for the design of a second generation of TPR modules with modified binding properties. Results Overall crystal structure The structure of the complex between the C-terminal Hsp90 peptide and the designed TPR module CTPR390 was refined to an R-value of 27.1% (free R-value 28.2%), using all reflections between 30 A ˚ and 2.85 A ˚ resolution (Tables 1 & 2). The crystallographic asymmetric unit (ASU) contains five monomeric CTPR390 molecules with one 5-mer peptide (MEEVD) bound in the concave cleft of each TPR subunit (Fig. 1A). The stereochemical parameters of the refined model are good (Table 2), with 98.2% of all nongly- cine residues located in the ‘most favorable’ region and the remaining 1.8% nonglycine residues located in the ‘additionally allowed’ regions of the Ramachandran plot. Crystal packing – head-to-tail packing The parent protein, CTPR3, crystallized as a monomer with two molecules in the ASU. It was therefore some- what surprising to find that CTPR390 forms ordered superhelical structures in the crystal (Fig. 1B–D). A superhelical arrangement has been previously observed in the crystal forms of CTPR8 and CTPR20 [7]. The packing in CTPR390 crystals, however, is dif- ferent. The CTPR390 units stack head to tail and form continuous pseudoinfinite crystalline helical ‘fibers’, which are arranged in a hexagonal symmetry lattice (Fig. 2A,B). In the CTPR8 and CTPR20 crystal forms, the ASU was composed of only part of the molecule (two or four repeats), so the ends of the molecules could not be located in the electron density map, and the full-length structures were reconstructed by apply- ing crystal symmetry and unit cell translations [7]. By contrast, with CTPR390, we observed five molecules in the ASU, and the discontinuity in the electron density that defines the end of each CTPR390 molecule was clear, allowing us to place the five individual units in the ASU (Fig. 1B,D). Each CTPR390 unit is com- posed of three TPR repeats (AB-helix pair) and an additional C-terminal capping helix (A cap ). The only way for molecules AB–AB–AB–A cap to arrange on ‘head-to-tail’ packing is if the C-terminal A cap -helix is displaced to allow B3–A1¢ intermolecular packing. This effect was observed previously in the CTPR8 and CTPR20 crystal forms [7]. CTPR390 superhelix – comparison with long TPR arrays The superhelical pitch for the CTPR390 superhelix is approximately 56 A ˚ , the diameter is 41.4 A ˚ , and the superhelical twist is 51.4°. Seven repeats form an almost complete superhelical turn (Fig. 3A,B). CTPR8 and CTPR20 structures displayed similar supehelical conformations, but with eight repeats per superhelical Table 1. X-ray data collection statistics. CTPR390–Hsp90 Space group R3 Unit cell dimensions a = b = 100.67 A ˚ , c = 161.57 A ˚ Wavelength (A ˚ ) 1.1001 Resolution (A ˚ ) 50–2.85 (2.95–2.85) R merge (%) a 7.5 (39.7) I ⁄ rI a 21.18 (1.16) Completeness (%) a 99.4 (99.7) Redundancy a 5.18 (5.28) v 2a 1.180 (0.928) Total reflections 28 406 Unique reflections 13 180 a Values in parentheses correspond to the highest-resolution bin. Table 2. Model refinement statistics. CTPR390–Hsp90 Resolution (A ˚ ) 30–2.85 R work ⁄ R free 27.1 ⁄ 28.2 Number of atoms 4440 Protein ⁄ ligand atoms 4422 Solvent atoms 18 Average B-factor 35.28 Average B-factor peptide 102.95 rmsd bond length (A ˚ ) 0.005 rmsd angles (°) 0.708 Ramachandran plot (% most favored) 98.2 A. L. Cortajarena et al. Structure of designed TPR module–ligand complex FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS 1059 turn and therefore a twist of 45°, with pitch values varying from 67 A ˚ to 72 A ˚ and diameter varying from 38 A ˚ to 42 A ˚ between different crystal forms [7]. We have previously published a detailed comparison of the superhelices formed by the CTPR proteins and the superhelix formed by the TPR domain of the enzyme O-linked GlcNAc transferase [8], showing that the two superhelices are similar [7]. The superhelix in CTPR390, even though it is similar to that previously observed in CTPR8 and CTPR20, is more compressed, and presents a larger curvature, with one fewer repeat per superhelical turn. These differences are clear when the first three repeats of the CTPR390 superhelix are superimposed onto the three N-terminal repeats of CTPR8, as shown in Fig. 3C. The N-terminal repeats align well, with an rmsd value of 0.897 A ˚ , but because of the differences in the superhelical twist, the two structures differ more and they do not overlap well towards the C-terminal repeats. The fact that 3-TPR units from CTPR390 align well with 3-TPR units of CTPR8 or CTPR20 indicates that, rather than the inter-repeat packing, the intermolecular packing is probably responsible for the pitch and diameter differ- ences between the two structures. Structure of individual CTPR390 molecules Considering the individual 3-TPR units, the structure of CTPR390 is almost identical to the structure of the parent protein, CTPR3 [3]. CTPR3 is the consensus protein, which contains no binding residues. CTPR390 has Hsp90-specific residues ‘grafted’ onto the binding surface of CTPR3 [5]. The pairwise backbone align- ment of CTPR3 (Protein Data Bank ID: 1Na0) and A B Fig. 2. Crystal packing of CTPR390. (A) R3 crystal lattice in the XY plane. The crystal axes (x, y, z) and the positions of the three-fold symmetry operators (black triangles) are indicated. The yellow box represents the unit cell. The arrows indicate the long axis of the crystalline superhelices. (B) Axial view of the crystalline superhelic- es in hexagonal arrangement. For simplicity, only the superhelices running in one direction in the crystal are shown to depict the hex- agonal symmetry. The crystal axes (x, y, z) are indicated. The yellow box represents the unit cell. ADBE C C E C A B D E A B D A C-termini N-termini A C D B Fig. 1. Crystal structure of CTPR390–Hsp90 peptide complex. (A) The ASU is shown in ribbon representation, with each CTPR390 unit colored differently (chain A, green; chain B, cyan; chain C, magenta; chain D, yellow; chain E, orange). The chains are labeled in the figure with their identification letters. The five Hsp90 peptide ligands (G, H, I, J, and K) are shown as gray ribbons. (B) Ribbon representation of a superhelix formed by five CTPR390 subunits, reconstructed by applying crystal symmetry and unit cell transla- tions. The color code for the different CTPR390 chains is the same as in (A). (C) Axial view of the superhelix in (B). (D) Schematic rep- resentation of the CTPR390 subunits packing in the infinite supe- rhelices in the crystal form [same color code as in (A–C)]. Structure of designed TPR module–ligand complex A. L. Cortajarena et al. 1060 FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS CTPR390 has an rmsd value of 0.738 A ˚ (Fig. 3D). When we calculate pairwise alignments of CTPR390 molecules within the ASU, we obtain rmsd values in the range 0.433–0.682 A ˚ , only slightly smaller than the values observed for the CTPR390–CTPR3 compari- son. The conformation of CTPR390 with the Hsp90 peptide ligand bound is thus very similar to the ligand-free CTPR3 structure. This result lends strong support to our hypothesis that CTPR3 is a stable framework onto which we can introduce mutations to change the binding specificity without affecting the structure of the protein. In addition, this result con- firms our previous observation that TPR modules undergo little or no structural change upon ligand binding [4]. CTPR390–peptide complex CTPR390 binds specifically and with moderate affinity (K d of 200 lm) to the C-terminal peptide of Hsp90 [5]. CTPR390 accommodates the Hsp90 peptide in its con- cave binding groove (Fig. 4A). The MEEVD peptide is in an extended conformation, very similar to that seen in the cocrystal structure of the TPR2A–MEEVD pep- tide complex (Protein Data Bank ID: 1elr) [9]. TPR2A is a natural Hsp90-binding TPR from Hsp-organizing protein. The resolution of the structure that we present is only 2.85 A ˚ , and when the structure was refined with no peptides modeled, electron density in the binding pockets of all five TPR units in the ASU was clearly evident. The Hsp90 peptide was built into this density, starting with the peptide from the TPR2A–Hsp90 com- AB CD Fig. 3. CTPR390 superhelix. (A) Molecular surface representation of one superhelical turn of CTPR390 (green) and CTPR8 (blue); the dimensions of the superhelices are shown. (B) Molecular surface representation of the CTPR390 superhelix in axial view. The diame- ter of the superhelix is shown. (C) Overlay of CTPR390 (green rib- bon) and CTPR8 (blue ribbon) superhelices. Backbone alignment of the first three N-terminal repeats of CTPR8 and CTPR390 chain C. The N-termini and C-termini of the superhelices are labeled. The A-helix and B-helix of the first repeat are also labeled. (D) Pairwise alignment of the CTPR390 structure (chain C in magenta) and the CTPR3 structure (Protein Data Bank ID: 1Na0 in blue). The N-ter- mini and C-termini of the proteins and the A-helices and B-helices of the three repeats are labeled. AB CD Fig. 4. X-ray crystal structure of CTPR390 in complex with the C-terminal peptide of Hsp90. (A) CTPR390–Hsp90 complex (protein chain C and peptide chain I). The backbone of CTPR390 is shown as a ribbon representation, and the side chains of the TPR residues, which directly interact with the peptide, are displayed as yellow sticks. The C-terminal Hsp90 peptide is shown as sticks in purple. (B) 2F o – F o electron density maps for two of the peptide chains in the ASU: peptide chain I. (C) Overlay of the five peptide chains (G, H, I, J and K chains). The peptide backbones are aligned, giving an rmsd value of 0.298 A ˚ . (D) Overlay of two peptide chains (I in magenta, and J in yellow) bound to two CTPR390 molecules in the ASU (C and D, respectively). The two views are related by 90° rota- tion about a vertical (y) axis. Only the protein chain backbones, and not the peptide chains, were overlayed, giving an rmsd value of 0.441 A ˚ . A. L. Cortajarena et al. Structure of designed TPR module–ligand complex FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS 1061 plex [9]. The 2F o – F c electron density map for one peptide in the asymmetric unit (Fig. 4B, peptide I) shows that the peptide is well defined in the complex. Positional noncrystallographic symmetry constraints between the five peptide chains in the ASU were used during the refinement. In the final stages of the refine- ment, the noncrystallographic symmetry constraints were released, and the five peptide chains in the ASU adopted slightly different locations relative to the TPR domains. When the backbones of the peptides are aligned, the five peptide molecules show almost identi- cal conformations, giving an rmsd value of 0.298 A ˚ (Fig. 4C). On the other hand, when the backbones of their corresponding TPR protein chains are overlayed, the average rmsd value of the pairwise peptide chains is 3.172 A ˚ . Figure 4D shows two views, related by 90° rotation, of the overlay of peptide chains I and J, and illustrates the conformational variability between the different peptide chains relative to the TPR domains. This result implies that the peptide chains can reorient as rigid bodies in the binding pocket. The average B-factor for atoms in the peptides is higher than the average B-factor for atoms in the protein (Table 2), which again may be a reflection of the mobility of the peptide chain in the binding pocket. This conforma- tional variability could exist because not all of the TPR–peptide interactions that are seen in the TPR2A– peptide complex are reproduced in the CTPR3–peptide complex. Such interactions are discussed in detail in the next section [5]. Atomic details of the CTPR390–Hsp90 interaction Analysis of the detailed interactions in the CTPR390– Hsp90 complex is presented for one of the complexes in the ASU: chains C (TPR) and I (peptide) (Fig. 4A), for which the electron density for the peptide is the clearest, and the confidence in the conformation of the peptide within the complex is the highest. The dissociation constant for the CTPR390–MEE- VD interaction is  200 lm [5], whereas the dissocia- tion constant of the TPR2A–MEEVD interaction  11 lm [9]. A comparison of the cocrystal structures of TPR2A and CTPR390 in complex with the MEE- VD peptide provides an explanation for the lower affinity of the designed protein. The backbone overlay of CTPR390 and TPR2A protein chains gives an rmsd value of 1.632 A ˚ , and shows that there are no large differences in the arrangement of the conserved peptide-binding residues. Rather, the major differences in the complexes are in the location of the peptide chains relative to the pro- tein (Fig. 5A). The Hsp90 peptide is located in the CTPR390 concave cleft further away from the protein than it is in the TPR2A domain. Accordingly, in the TPR2A–Hsp90 complex, there are more extensive and closer interactions between the protein side chains and the peptide, which probably contribute to the tighter binding affinity. We analyzed in detail the interactions present in the designed complex, in which we introduced Hsp90- specific binding residues, mimicking the TPR2A binding interface. Consequently, we expected to find in the CTPR390 protein interactions comparable to those present in the naturally occurring Hsp90-binding TPR domains. A large energetic contribution to the binding of the peptide MEEVD to TPR2A comes from interactions of the EEVD motif with five conserved ‘carboxylate clamp’ residues on the binding face of the TPR [9]. CTPR390 was generated by grafting these residues, and three additional Hsp90-binding specific residues, onto its concave binding face. The five residues that form the carboxylate clamp in CTPR390 (Lys13, Asn17, Asn48, Lys78, and Arg82) are equivalent to the residues in TPR2A (Lys229, Asn233, Asn264, Lys301, and Arg305). The overlay of the Ca atoms of these five binding residues gives an rmsd value of 0.565 A ˚ (Fig. 5A), as compared with the rmsd value of 1.632 A ˚ when the entire TPR domains are aligned. When the ‘clamp residues’ are aligned, the superposition of the two Hsp90 peptides shows that the C-terminal residues of the peptides align reasonably well and present the same overall conformation. At the N-terminus of the peptide, the alignment diverges more, with the major difference being that the N-terminal Met is signifi- cantly further away from the binding cleft in the CTPR390–MEEVD complex than in the TPR2A– MEEVD complex (Fig. 5A). Figure 5B,C shows detailed schematic diagrams of the TPR–ligand interactions for CTPR390 and TPR2A, respectively, generated using ligplot [10]. The electrostatic interactions and hydrogen-bonding inter- actions mediated by the conserved carboxylate clamp residues for the TPR2A–Hsp90 and CTPR390–Hsp90 complexes are tabulated and compared in Table 3. The CTPR390–Hsp90 complex reproduces most of the key interactions present in the TPR2A–Hsp90 complex. In the CTPR390–Hsp90 structure, the water molecules cannot be located clearly, so the interactions present in the TPR2A–Hsp90 complex mediated by water molecules could not be placed in the CTPR390– Hsp90 complex (which does not mean that they are not present). Additionally, for most of the interactions, the distances between the interacting atoms are greater in the CTPR390–Hsp90 complex than in the TPR2A– Structure of designed TPR module–ligand complex A. L. Cortajarena et al. 1062 FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS Hsp90 complex (Table 3), which could explain the weaker binding affinity relative to the TPR2A–Hsp90 domain. In addition to the electrostatic interactions, hydro- phobic interactions also contribute to the TPR–peptide affinity. The total surface area buried upon complex formation between the TPR and the MEEVD peptide was calculated using getarea [11]. In the CTPR390– MEEVD complex, 810 A ˚ 2 of surface area is buried upon complex formation, which is slightly smaller than the surface area buried in the TPR2A–Hsp90 complex (930 A ˚ 2 ) [9]. The hydrophobic residue Val4 of the Hsp90 peptide is accommodated in a hydrophobic pocket formed by Asn233, Asn264 and Ala267 in TPR2A. A comparable hydrophobic pocket is formed by Asn17, Tyr20, Asn48 and Asn51 in CTPR390 (Fig. 5B,C). In these two cases, the total surface area buried upon binding of the Val is virtually identical (128 A ˚ 2 versus 137 A ˚ 2 ). Met1 of the Hsp90 peptide is also engaged in tight hydrophobic interactions with a cavity mainly formed by the side chains of Tyr236 and Glu271 of TPR2A (Fig. 5C). However, in the CTPR390–Hsp90 complex, although an equivalent Tyr is present (Tyr55), there is a Lys (Lys55) at the Glu271 position that pushes the Met outside of the binding pocket. Therefore, Met does not contribute to the binding, resulting in a weaker binding affinity (Fig. 5A,B). A comparison of the average B-factors for the resi- dues in the MEEVD peptide show that the C-terminal Asp has a B-factor of 84, whereas the N-terminal Met has a B-factor of 125. These values provide additional support for the notion that the Met is not engaged in specific interactions with the protein. Therefore, the Met probably has more conformational flexibility than A B C Fig. 5. CTPR390–Hsp90 interactions and comparison with the TPR2A–Hsp90 complex. (A) Overlay of the five carboxylate clamp residues of the CTPR390–Hsp90 (magenta) and TPR2A–Hsp90 (blue) complexes. The side chains of the protein residues and the two Hsp90 peptides are shown in stick representation. The identi- ties of the residues in both the CTPR390 (top) and TPR2A (bottom) domains and the N-termini and C-termini of the peptides are indi- cated. (B) Schematic 2D diagram of CTPR390–Hsp90 peptide inter- actions (chains C and I). The schematic was generated from the pdb file of the complex with LIGPLOT [10]. The interactions shown are those mediated by hydrogen bonds and by hydrophobic con- tacts. Hydrogen bonds are indicated by dashed lines between the atoms involved, and hydrophobic contacts are represented by an arc with spokes radiating towards the ligand atoms that they con- tact. The contacted atoms are shown with spokes radiating back. (C) Schematic representation of TPR2A–Hsp90 peptide interactions generated as in (B) from the pdb file of the complex (Protein Data Bank ID: 1elr) [9]. A. L. Cortajarena et al. Structure of designed TPR module–ligand complex FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS 1063 the Asp, which displays many specific contacts with the protein (Table 3). The change in surface area associated with the Met upon binding to CTPR390 (34 A ˚ 2 )is small in comparison with the change upon binding to TPR2A (142 A ˚ 2 ), also corroborating the lack of specific interaction mediated by the Met. Indeed, the difference in surface area buried by the Met between the two complexes accounts for the total difference in surface area buried upon peptide binding between CTPR390 and TPR2A. It has been reported that deletion of the Met increases the dissociation constant of the TPR2A– Hsp90 complex from 11 lm to 90 lm [9]. Therefore, the lack of this interaction in the CTPR390–peptide complex will partially contribute to the moderately weak binding affinity of the designed TPR module. Discussion In this article, we present the cocrystal structure of a designed TPR domain with its partner peptide. We show that this 3-TPR domain can adopt a superhelical structure in the crystal similar to those reported for long TPR arrays [7]. This result illustrates the natural tendency of TPR domains to stack head to tail and self-assemble into an ordered macrostructure in crystals. We have seen no evidence for such associa- tion in solution. We previously showed that, by grafting the binding residues from a given natural TPR domain onto a con- sensus scaffold, we could incorporate the binding activity in the newly designed domain. This structure proves that the new domain obtained using this ‘graft- ing’ strategy mimics not only the binding activity [5,6], but also the interactions at a molecular level between the protein and the ligand. This result confirms the TPR domains as a stable protein scaffold where, by grafting the binding residues, one can interchange the binding activities between domains. Additionally, this work allows us to compare the structure of the consensus CTPR3 domain without ligand and the designed CTPR390 (with a total of only 12 mutations relative to the parent CTPR3) with ligand bound. These two structures overlap almost per- fectly, supporting our previous observations that TPR domains bind their target peptides without undergoing any major conformational changes [4]. Finally, the detailed understanding of the molecular basis of the CTPR390–Hsp90 recognition opens the door to a second generation of rationally improved CTPR modules. For example, it is clear from the structure that Asn51 and Lys55 from CTPR390 are pushing the peptide out of the hydrophobic pocket, and therefore the N-terminal Met of the peptide does not contribute to the binding energy. In TPR2A, these residues are Ala267 and Glu271. One would expect that introducing these mutations in the CTPR390 scaf- fold might improve its binding affinity for Hsp90 peptide. Experimental procedures Protein design CTPR390 incorporates Hsp90-binding residues in the con- cave face of the consensus 3-TPR domain (CTPR3) [3,5]. The sequences of the first, second and third A-helices of CTPR390 are as follows: first A-helix, AEAWKNLGNAYYK; second A-helix, ASAWYNLGNAYYK; and third A-helix, AKA- WYRRGNAYYK. The B-helix sequence in all of the TPR repeats in CTPR390 is DYQKAIEYYQKALEL, which differs from the negatively charged back sequence of the Table 3. TPR–peptide electrostatic interactions in the carboxylate clamp. For the data for hydrogen-bonding interactions, we have been gen- erous in the constraints in order to show all the possible interactions, and how they differ between the two complexes. TPR2A–Hsp90 interactions CTPR390–Hsp90 interactions Residue in TPR Residue in peptide Distance (A ˚ ) Residue in TPR Residue in peptide Distance (A ˚ ) K229 D5 (OXT) 2.68 K13 D5 (OXT) 3.09 N233 D5 (OXT) 2.83 N17 D5 (OXT) 3.99 N264 D5 (OXT) 2.83 N48 D5 (OXT) 2.94 D5 (NH) 2.96 D5 (NH) 3.18 H 2 O–D5 (OD2) 2.68–3.03 – – K301 D5 (OD1) 2.63 K78 D5 (OD1) 2.87 D5 (OD2) 3.04 R305 E3 (O) 2.73 R82 E3 (O) 2.64 H 2 O–E3 (NH) 3.13–2.71 – – E2 (OE1) 2.78 E2 (OE1) 2.79 E2 (OE1) 3.10 Structure of designed TPR module–ligand complex A. L. Cortajarena et al. 1064 FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS CTPR3 scaffold, DY DEAIEYYQKALEL. Underlining indi- cates the solvent-exposed charged residues [5]. Cloning of the CTPR390 gene The gene encoding CTPR390 was constructed as previously described and cloned into the pProEx-HTA vector to incor- porate a cleavable N-terminal His-tag (GibcoBRL, Gaithersburg, MD, USA) [5,12]. The identity of the construct was verified by DNA sequencing (W.M. Keck Facility, Yale University, New Haven, CT, USA). Protein expression and purification CTPR390 was overexpressed and purified as previously described [5]. As a final purification step to obtain high- purity protein for crystallization, the protein was run on a size exclusion column (HiLoad Superdex HR-75; Amer- sham Bioscience, Uppsala, Sweden). The protein concentra- tion was determined by UV absorbance at 280 nm, using extinction coefficients at 280 nm calculated from amino acid composition [13]. Protein crystallization and data collection Purified CTPR390 at 20 mgÆmL )1 protein in 10 mm Tris ⁄ HCl and 50 mm NaCl (pH 7.5) was mixed with the C-terminal five amino acids of Hsp90 (Ac-MEEVD-COOH peptide) at a protein ⁄ peptide ratio of 1 : 4. Microbatch- under-oil screening at the high-throughput crystallization laboratory at the Hauptman-Woodward Medical Research Institute Inc. (HWI, Buffalo, NY, USA) [14] identified few preliminary crystallization conditions. We could reproduce one crystallization condition [0.1 m NaH 2 PO 4 , 40% (w ⁄ v) poly(ethylene glycol) 20000, 0.1 m Caps, pH 10.0] in our laboratory. We optimized this condition by the sitting-drop vapor diffusion method, using two-fold diluted initial for- mulation as the well solution. The final crystallization condi- tion contained 50 mm NaH 2 PO 4 , 20% (w ⁄ v) poly(ethylene glycol) 20000, and 50 mm Caps (pH 10.0). The well solution was mixed in equal volumes (2 lL) with a protein–peptide complex solution (1 : 4 molar ratio) at 30 mgÆmL )1 protein concentration. Crystals appeared within a week at 20 °C, and reached sizes of approximately 80 · 80 · 50 lm within 2 weeks. Crystals were flash-cooled under a nitrogen gas stream (100 K). Data were collected to 2.85 A ˚ resolution at the NSLS beamline X12C (Brookhaven National Labora- tory). The data collection statistics are shown in Table 1. Structure determination and refinement We used hkl2000 [15] to index, scale and integrate the data. The protein crystallized in space group R3 with unit cell dimensions of a = b = 100.67 A ˚ , c = 161.57 A ˚ , and a = b =90°, c = 120°. The CTPR390 structure was solved by molecular replacement using molrep [16] in the ccp4i suite [17]. The structure of the consensus TPR with- out the solvating helix was used as search model [CTPR3 (Protein Data Bank ID: 1NA0] [3]. There were five TPR molecules in the ASU. The structure was refined with cns [18] and refmac5 [19], with TLS refinement [20] in the late stages of the refinement, to a resolution of 2.85 A ˚ . Iterative rounds of refinement and manual model adjusting in coot [21] were performed until R-factors converged to a final value of R (R free ) = 28.4 (29.2) for the structure of the TPR molecules. The ligand peptide (MEEVD) was built in the F o –F c difference electron density map. First, one peptide chain was built in the CTPR390 molecule in the ASU with strongest positive density, using a backbone conformation for the Hsp90 peptide from the TPR2A–Hsp90 complex as starting model (Protein Data Bank ID: 1elr) [9]. The model with one copy of the Hsp90 peptide was refined and the additional four peptide chains were built by symmetry oper- ations of the refined peptide chain in the binding pockets of the other protein chains. The complete model was further refined. Water molecules were automatically added in coot, and were validated with the electron density maps. The final model with one peptide molecule in the binding groove of each of the five TPR molecules in the ASU converged to R (R free ) = 27.1 (28.2). The geometry and stereochemical properties of the model were checked with molprobity [22]. Crystallographic statistics are shown in Table 2. Coordinates The X-ray structure of the CTPR390–Hsp90 peptide com- plex has been deposited in the Protein Data Bank as 3KD7. Acknowledgements We thank members of staff at NSLS beamlines X12C and X6A, BNL, where data were collected. The high- throughput crystal screening service of the Hauptman- Woodward facility assisted in identifying initial crystallization conditions. We thank T. Kajander for his advice during the crystallization process and data collection. 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Acta Crystallogr D 60, 2126–2132. 22 Lovell SC, Davis IW, Arendall WBI, de Bakker PIW, Word JM, Prisant MG, Richardson JS & Richardson DC (2003) Structure validation by C-alpha geometry: phi, psi, and C-beta deviation. Protein Struct Funct Genet 50, 437–450. Structure of designed TPR module–ligand complex A. L. Cortajarena et al. 1066 FEBS Journal 277 (2010) 1058–1066 ª 2010 The Authors Journal compilation ª 2010 FEBS . Crystal structure of a designed tetratricopeptide repeat module in complex with its peptide ligand Aitziber L. Cortajarena 1 , Jimin Wang 1 and Lynne Regan 1,2 1 Department of Molecular Biophysics. three TPR repeats (AB-helix pair) and an additional C-terminal capping helix (A cap ). The only way for molecules AB–AB–AB A cap to arrange on ‘head-to-tail’ packing is if the C-terminal A cap -helix. we created a protein (CTPR390) that incorporates heat shock protein (Hsp)90-binding residues, grafted from natural Hsp90-binding TPR domains, onto the con- cave ligand- binding face of the domain (A- helices)