Protein design to understand peptide ligand recognition by tetratricopeptide repeat proteins

Aitziber L. Cortajarena1, Tommi Kajander1, Weilan Pan1, Melanie J. Cocco1,2 and Lynne Regan1,3,4

1Department of Molecular Biophysics and Biochemistry and 3Department of Chemistry, Yale University, PO Box 208114, New Haven, CT 06520-8114, USA 2Present address: Department of Molecular Biology and Biochemistry, University of California, 1218 Natural Sciences I, Irvine, CA 92697-3900 USA

4 To whom correspondence should be addressed. E-mail: lynne.regan{at}yale.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Protein design aims to understand the fundamentals of protein structure by creating novel proteins with pre-specified folds. An equally important goal is to understand protein function by creating novel proteins with pre-specified activities. Here we describe the design and characterization of a tetratricopeptide (TPR) protein, which binds to the C-terminal peptide of the eukaryotic chaperone Hsp90. The design emphasizes the importance of both direct, short-range protein–peptide interactions and of long-range electrostatic optimization. We demonstrate that the designed protein binds specifically to the desired peptide and discriminates between it and the similar C-terminal peptide of Hsp70.

Keywords: consensus sequence/electrostatics/Hsp90/protein design/tetratricopeptide repeat


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
A central goal of protein design is to create proteins with novel binding specificities. It is therefore of key importance to understand the determinants of affinity and specificity in protein–ligand interactions. Such understanding facilitates the design of proteins that are able to bind to a ligand of choice. One can envision a wide range of potential applications for such designer proteins, from diagnostics to the manipulation of cellular function in either an analytical or therapeutic fashion. A number of design strategies have been used for generating DNA or protein-binding proteins based on computational methods, combinatorial libraries or rational design (Pabo et al., 2001Go; Reina et al., 2002Go; Kohl et al., 2003Go).

Within the cell there are a vast number of protein–protein interactions, which range in affinity from extremely tight, essentially permanent complexes [the ribosome, for example (Ban et al., 2000Go)] to more transient interactions that serve to bring a particular protein into the right place at the right time [the interaction of Hop with Hsp90 and Hsp70, for example (Hernandez et al., 2002Go; Wegele et al., 2004Go)].

Non-globular, repeat proteins are widely used to mediate protein–protein interactions (Andrade et al., 2001aGo). Repeat proteins are constructed from a variable number of similar structural motifs arrayed in tandem (Groves and Barford, 1999Go; Kobe and Kajava, 2000Go; Andrade et al., 2001aGo; Kajava, 2001Go). They typically have large exposed interaction surfaces, which may explain their functional prevalence as mediators of protein–protein interactions. Examples include ankyrins (ank) (Sedgwick and Smerdon, 1999Go), armadillo repeats (ARM) (Hatzfeld, 1999Go; Coates, 2003Go), HEAT repeats (Andrade et al., 2001bGo), hexapeptide repeats (HPR) (Jenkins and Pickersgill, 2001Go), leucine-rich repeats (LRR) (Kobe and Deisenhofer, 1994Go, 1995Go) and tertratricopeptide repeat (TPR) motifs (D'Andrea and Regan, 2003Go; Main et al., 2003aGo).

The wide variety of interaction surfaces formed by different types of repeat proteins is reflected in the diverse binding specificities that they exhibit (Baumann et al., 1993Go; Jacobs and Harrison, 1998Go; Scheufler et al., 2000Go). Furthermore, although proteins in the same repeat family may bind their target molecules in the same general fashion, they may each exhibit different and unique ligand binding specificities (Blatch and Lassle, 1999Go; Kobe and Kajava, 2001Go).

The tetratricopeptide repeat (TPR) was identified and named as a 34 amino acid degenerate sequence that occurs in tandem repeats in a variety of proteins (Hirano et al., 1990Go; Sikorski et al., 1990Go; Lamb et al., 1995Go). Since the original description, TPR domains have been identified in many different proteins and are implicated in a wide variety of cellular functions. Different TPR domains exhibit different protein binding specificities and function to mediate protein–protein interactions (Blatch and Lassle, 1999Go). Interactions of this type may also facilitate assembly of the TPR-protein into higher order complexes (Gatto et al., 2000Go; Lapouge et al., 2000Go).

The three-dimensional structure of a single TPR is a helix–turn–helix; adjacent TPR repeats stack in parallel and form a right-handed superhelix (Das et al., 1998Go). Although the number of tandem repeats identified at the sequence level varies from one to 16, three tandem repeats is the most prevalent length and may represent the minimal number of repeats required for specific ligand binding (D'Andrea and Regan, 2003Go), though a one-TPR structure has been reported (Abe et al., 2000Go). It is perhaps not surprising, therefore, that certain three-TPR motifs are the best characterized, both structurally and functionally. For most TPR domains, however, neither the structure, the ligand nor the mode of ligand binding have yet been determined.

High-resolution crystal structures of five three-TPR domains, have been reported, two in complexes with their peptide ligand (Scheufler et al., 2000Go) and three as free protein (Das et al., 1998Go; Taylor et al., 2001Go; Sinars et al., 2003Go). When we compared these structures, we found that the different TPR structures are virtually superimposable with backbone r.m.s.d. values that vary from 1.1 to 1.9 Å for different pairwise structural alignments [carried out using the molecular graphics software O (Jones et al., 1991Go)]. There are no consistent differences between the structures of free TPR domains and the structures of those in complex with a peptide ligand. Crystal structures of three-TPR domains in complex with their peptide ligands reveal that the target peptide is bound in an extended conformation on the front/concave binding surface of the TPR superhelix (Gatto et al., 2000Go; Scheufler et al., 2000Go). The molecular interaction of Hop TPR domains, TPR1 and TPR2A with the C-terminal peptides of proteins Hsp70 and Hsp90, respectively, are particularly well characterized and provide a good starting point for understanding TPR-target recognition (Scheufler et al., 2000Go).

We recently described the design of a consensus TPR sequence, in which the global propensity of each amino acid occurring at each position was calculated from an analysis of all TPR sequences in the Pfam database, such that any differences arising from different binding specificities is averaged out and structurally important features dominate (D'Andrea and Regan, 2003Go; Main et al., 2003bGo). Complementing the original structural analysis of the natural three-TPR domain from PP5 (Das et al., 1998Go), our studies revealed a conserved pattern, at a few key positions, of small and large hydrophobic residues that are sufficient for specifying the TPR fold.

In the original study we investigated how the number of tandem repeats of the consensus TPR sequence influences protein structure and stability. Here we describe further designs, in which we use the protein with three tandem consensus TPR repeats (CTPR3) as a minimal sized, stable structural scaffold on which to introduce specific binding functionality.

A wide range of different binding specificities can be accommodated on a three-TPR scaffold, but in this first instance we chose to introduce an activity that is well characterized in a natural three-TPR domain: binding to the C-terminal peptide of Hsp90. This approach allows us to dissect the contributions of different interactions to binding affinity and specificity and provides information that is complementary to studies in which natural proteins are mutated.

Hsp90–TPR interactions are of intrinsic biological interest, because the activity of Hsp90 is dependent on its interaction with TPR-containing proteins (Johnson et al., 1998Go; Prodromou et al., 1999Go). Furthermore, overexpression of Hsp90-binding TPR domains has a dominant negative effect in vivo (Chang et al., 1997Go) and inhibition of Hsp90 activity is being pursued as a possible route to novel anti-cancer agents (Neckers and Schulte, 1999Go; Bisht et al., 2003Go; Workman, 2003Go).


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Statistical analysis of Hsp90-binding TPR sequences

The framework for the functional design was CTPR3, a stable, regular, three-TPR protein. Each of the three TPR repeats in CTPR3 is identical and represents the consensus sequence derived from all TPR sequences identified in Pfam (1837 sequences in 2002, http://pfam.wustl.edu) (Main et al., 2003bGo). Thus, CTPR3 is optimized for TPR structural features, but not for any specific ligand binding activity.

There is more than one three-TPR protein that binds to Hsp90 by a specific interaction with the five C-terminal amino acids of the chaperone. We performed an alignment of nine TPR sequences from non-homologous proteins that bind to Hsp90 (these were human HopTPR2A domain, PP5, FKBP51, FKBP52, Cyp40 and Tom70 and yeast Crp6, Cyp7 and CNS1). For each of the three TPR repeats we compared the frequency of occurrence of different amino acids at each of the 34 positions in a TPR. Positions at which there was a clear preference for a particular amino acid in the Hsp90-binding TPRs subset, but not in TPRs as a whole where incorporated into the functional design.

The two proteins that were designed for the binding studies reported here are CTPR390 (which incorporates amino acids involved in peptide binding on the front/concave surface and has an electrostatically optimized/neutralized back/convex surface) and CTPR390-B (which incorporates only the amino acids involved in peptide binding on the front/concave surface and which has a negatively charged back/convex surface).

Cloning and molecular biology

Genes encoding the proteins were constructed by Klenow extension of six overlapping oligonucleotides to generate three double-stranded DNA fragments. Two rounds of PCR amplification were then performed, using primers complementary to each end, to generate large quantities of the full-length gene. The final product was digested with BamHI and HindIII and subcloned into the pProEx-HTA vector (Gibco, Gaithersburg, MD). The vector results in a gene that codes for a His6-tagged protein. The identity of each construct was confirmed by DNA sequencing (W.M. Keck Facility, Yale University, New Haven, CT).

The constructs we made produced the desired proteins with an N-terminal His6-tag followed by a TEV protease cleavage site, which allowed the His6-tag to be removed after affinity purification. After such cleavage, because of the vector used for cloning, the proteins all had a five amino acid (GAMDP) N-terminal extension.

Protein expression and purification

Proteins were expressed and purified based on a previously described protocol (Main et al., 2003bGo). Briefly, the plasmids were transformed into BL21(DE3) cells. Overnight cultures were used to inoculate (1:200 dilution) 1 l cultures of LB and grown at 37°C to an OD600 of 0.8, then induced with 0.6 mM IPTG and grown 5 h at 30°C. Cultures were centrifuged and the cell pellets were resuspended in 30 ml of 300 mM NaCl, 10% glycerol, 50 mM Tris, pH 8.0. One tablet of Complete EDTA-free protease inhibitor cocktail (Roche, Basel, Switzerland) and lysozyme to a final concentration of 1 mg/ml were added to this suspension. The suspension was incubated for 30 min on ice, then sonicated and the lysate cleared by centrifugation for 1 h at 35 000 g. The CTPR3 proteins were present in the supernatant after this spin and were purified by cobalt-based metal affinity chromatography using BD TALON metal affinity resin (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's instructions. The His6-tag was cleaved by overnight TEV protease digestion at room temperature. The cleaved His6-tag and the TEV protease (itself His6-tagged) were removed from the CTPR preparation by a metal affinity column. Size exclusion gel filtration chromatography through a HiLoad Superdex HR-75 column (Amersham Bioscience, Uppsala, Sweden) was performed as the final step of the purification. The final yield was about 40 mg/l of culture. All protein concentration determinations were performed by measuring the UV absorbance at 280 nm; the extinction coefficients at 280 nm were calculated from amino acid composition (Pace et al., 1995Go).

Circular dichroism (CD)

CD spectra were acquired in 150 mM NaCl, 50 mM phosphate, pH 6.3 buffer using an AVIV Model 215 CD spectrophotometer (AVIV Instruments, Lakewood, NJ). CD spectra were recorded at 6 µM protein concentration at 25°C in a 0.1 cm pathlength cuvette.

Thermal denaturation studies were performed at 6 µM protein concentration in the buffer specified above in a 0.1 cm pathlength cuvette. The ellipticity at 222 nm was recorded in the forward direction from 15 to 95°C and in the reverse direction cooled to 15°C in 1°C steps with an equilibration time of 1 min at each temperature. To calculate the melting temperature (Tm), the data were fitted to a two-state model transition.

The equilibrium stability was measured by Gu·HCl-induced denaturation, following the ellipticity at 222 nm. Solutions of the identical concentration of protein (3 µM) were prepared in either 150 mM NaCl, 50 mM phosphate, pH 6.3 buffer (folded protein) or 150 mM NaCl, 7 M Gu·HCl, 50 mM phosphate, pH 6.3 (unfolded protein). An automatic titration was performed, mixing increasing amounts of the denatured protein with folded protein such that the total protein concentration was held constant but the Gu·HCl concentration increased in 0.2 M intervals. Measurements were made in a 1 cm pathlength cuvette thermostated at 25°C with equilibration times of 45 min at each guanidinium concentration.

Data were evaluated for thermodynamic parameters by fitting to a two-state unfolding model with the following equation (Clarke and Fersht, 1993Go):

where F is the spectroscopic signal, in this case ellipticity at 222nm, {alpha}N and {alpha}D are linear extrapolated y intercepts and ßN and ßD are the slopes of the baselines at the native (N) and denatured (D) states, [D] is the denaturant concentration, [D]50% is the denaturant concentration at which 50% of the protein is denatured, m is the slope of the transition, T is the temperature in kelvin and R is the gas constant (1.98 x 10–3 kcal/K·mol). Solving the equation by curve-fitting gives [D]50% and m values. The stability in the absence of denaturant ({Delta}GH2O) was determined using the equation

Molecular modeling and electrostatics calculations

To construct a model for the TPR–peptide complexes, the crystal structures of CTPR3 and of HopTPR2A in complex with its peptide ligand were aligned through their backbone C{alpha} atoms, using the graphics software O (Jones et al., 1991Go). The Hsp90–MEEVD peptide was then docked on to the CTPR390 binding groove, in an orientation corresponding to that observed in the HopTPR2A–peptide complex crystal structure (Figure 2) (Scheufler et al., 2000Go).



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Fig. 2. Surface representation of the electrostatic potential of the front/concave (on the top) and back/convex (on the bottom) faces of TPR domains. Two natural Hsp90-binding TPRs, TPR2A domain of Hop with its peptide ligand bound and PP5 and the designed proteins CTPR3 and CTPR390, are shown. Color range deep red to deep blue corresponds to the range in values of electrostatic potential from –16 to +20 kT/e, where k is Boltzmann's constant, T is absolute temperature and e is a proton's charge. The figures were created using GRASP (http://trantor.bioc.columbia.edu/grasp/) (Nicholls et al., 1993Go).

 
The positions of the side chains of residues that were changed in CTPR390 from CTPR3 were modeled using one of the most frequent rotamer values (as represented in the rotamer library in O).

No further modeling or minimization of the complex was performed. Instead, the peptide was moved back from the binding site by ~2 Å. This procedure avoided the introduction of steric clashes and also obviated the need to model specific close contacts and atomic interactions. Using this strategy, we constructed models for both the CTPR390–MEEVD and CTPR390-B–MEEVD complexes. We judged it inappropriate to attempt to model in specific protein–peptide contacts, because we do not yet have the crystal structures of either CTPR390 or CTPR390-B.

CTPR390 has a charge neutralized back/convex surface whereas CTPR390-B has a negatively charged back/convex face. Because we generated these models in the same fashion, the only difference between the two models is the charged residue pattern on the back/convex face of the protein. Hydrogen atoms were added to these models with the WHATIF web interface (at http://www.cmbi.kun.nl/gv/servers/WIWWWI/) and these all-atom models were used for the electrostatics calculations. The CHARMM22 parameter set was used to specify charges and atomic radii, with all atoms carrying partial charges. Continuum electrostatic calculations were carried out with the program DelPhi (Gilson et al., 1987Go).

Calculations were carried out on a cubic grid with 200 x 200 x 200 grid points in two rounds. The first potential map was calculated with 30% fill and dipolar boundary conditions. Final calculations were performed with 80% fill, with focusing boundary conditions as implemented in DelPhi. Other parameters were set as follows: salt concentration was varied from 0 to 300 mM and protein dielectric constant was set as {varepsilon}(p) = 4 and solvent dielectric constant as {varepsilon}(s) = 80. In each calculation the electrostatic energy of binding was estimated from differences in the grid energies as {Delta}{Delta}Ebind = {Delta}Ecomplex{Delta}Eapo-protein{Delta}Epeptide (assuming that the protein and peptide behave as rigid bodies) and the difference, arising solely for back/convex face charge differences, was estimated as the difference in {Delta}{Delta}Ebind between CTPR390 and CTPR390-B. The apo-protein and peptide calculations were performed such that the position and grid spacing were identical for the molecules in all calculations.

NMR spectroscopy

NMR spectra were recorded on either a Varian Inova 800 MHz or a Unity Plus 600 MHz spectrometer, with 15N-labeled protein in 150 mM NaCl, 10% D2O, 50 mM phosphate, pH 6.3 at 20°C. 1H–15N HSQC spectra (800 MHz) were acquired at 500 µM protein concentration. In the peptide titration experiment, increasing amounts of the 20-mer C-terminal peptide of Hsp90 (H2N-TEEMPPLEGDDDTSRMEEVD-COOH) were added to the protein sample and HSQC spectra were recorded after an incubation time of 30 min at each peptide concentration; 600 MHz 1H–15N HSQC-NOESY ({tau}m = 100 ms) and 1H–15N HSQC-TOCSY ({tau}m = 55 ms) spectra were collected at 1.5 mM protein concentration.

CTPR390 NH cross peaks were assigned based on the assignments for CTPR2 and CTPR3 (M.J.Cocco, E.Main and L.Regan, unpublished data). The assignments were refined and confirmed with 1H–15N HSQC-NOESY and 1H–15N HSQC-TOCSY experiments.

All data were collected at 20°C and processed with NMRPipe (Delaglio et al., 1995Go) and analyzed with Sparky (Goddard and Kneller, 1995Go).

Surface plasmon resonance-based binding assays

Surface plasmon resonance (SPR) measurements were performed on a BIACORE 3000 system (BIACORE, Uppsala, Sweden). The chips were made by immobilization of neutravidin on a CM4 sensor chip, using standard amide coupling. Subsequently, 200–300 RU of biotinylated peptides at 200 µg/ml in HBS-EP buffer (150 mM NaCl, 3 mM EDTA, 0.005% v/v polysorbate 20, 10 mM HEPES, pH 7.5) were immobilized on the neutravidin chip. The N-terminally biotinylated 24-mer C-terminal peptides of Hsp90 (biotin-SAAVTEEMPPLEGDDDTSRMEEVD-COOH) and Hsp70 biotin-GGFPGGGAPPSGGASSGPTIEEVD-COOH) that we used were synthesized and purified by HPLC and their identities were verified by mass spectrometry at the W.M. Keck Facility, Yale University, New Haven, CT. In the binding experiments 24-mer peptides were used to extend the position of the C-terminus of the peptide further from the chip surface. It has been shown that peptides corresponding to the C-terminal five amino acids of Hsp90 and to the C-terminal seven amino acids of Hsp70 bind to the TPR2A and TPR1 domains of Hop, respectively, with the same affinity as full-length Hsp90 or Hsp70 (KD TPR2A {approx} 8.5 µM; KD TPR1 {approx} 17 µM) (Scheufler et al., 2000Go; Brinker et al., 2002Go). For binding studies, 40 µl of pure TPR protein solutions at various concentrations (Figures 4 and 7) were injected over immobilized peptides in HBS-EP buffer at a flow rate of 40 µl min–1 using the KINJECT mode (600 s dissociation time). Complete regeneration of the chip surfaces was achieved by two 40 µl injections of 1 M NaCl at the same flow rate using the QUICKINJECT injection type. Background binding to neutravidin was subtracted from each signal to account for non-specific binding.



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Fig. 4. (A) Interaction of CTPR3 (open circles) and CTPR390 (filled circles) with Hsp90 C-terminal 24mer peptide analyzed by SPR. (B) Interaction of CTPR390 with Hsp90 (filled circles) and Hsp70 (open triangles) C-terminal peptides. Equilibrium response levels (Req) versus protein concentration for binding to C-terminal 24mer peptides. The dissociation constants were calculated by fitting the data to a steady-state 1:1 binding model (KD CTPR390–Hsp90 = 200 µM).

 


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Fig. 7. Interaction of TPR domains with the amidated C-terminal peptide of Hsp90 determined by SPR. Binding of CTPR390 (A) and TPR2A domain of Hop (B) to 24mer C-terminal peptide of Hsp90 (filled circles) and to the 24merC-terminal peptide, CONH2 amidated peptide (open circles). Equilibrium responses (Req) were plotted against protein concentration.

 
To determine the dissociation constants (KD), the average equilibrium response values (Req) were plotted versus the protein concentration injected and the titration curve was fit to a one-site binding model using SigmaPlot (Systat Software, Point Richmond, CA) with the following equation:

where KD is the dissociation constant, [P] the protein concentration in the mobile phase and Rmax the equilibrium response at saturation.


    Results
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Design of an Hsp90 binding TPR

The two main components of the functional design were to build in specific protein–ligand contacts and to improve the overall electrostatic interactions of the protein with negatively charged ligands.

The designed three-TPR protein CTPR3 was chosen as the scaffold on which to introduce specific ligand binding activity for several reasons. First, three tandem consecutive TPR repeats is the most common length in all TPR proteins from all kingdoms and all known Hsp90-binding TPR domains have three-TPR repeats. Second, structural considerations suggest that the three-TPR unit may represent the minimum number of repeats needed to form a generally functional protein–protein interaction unit (D'Andrea and Regan, 2003Go). Finally, CTPR3 is significantly more stable than natural three-TPR domains.

Residues that are a common feature of only Hsp90-binding TPRs were identified by comparing the frequency of occurrence of each amino acid, at each of the 34 positions of the repeat, for all the TPR sequences in the Pfam database with the sequences of only those TPR domains that bind to the C-terminal peptide of Hsp90 (Figure 1A). In contrast to the scaffold design, where the statistical analysis was carried out on a one-TPR repeat, for the functional design each of the three-TPR repeats were analyzed because the crystal structures of natural TPRs with their ligand suggest three-TPR repeats as a binding unit.



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Fig. 1. Statistical comparison analysis of all TPRs and Hsp90-binding TPRs. (A) The x-axis shows the different amino acids (one-letter code). The y-axis shows the frequency at which that amino acid occurs in all TPR motifs (red), the first (orange), second (yellow) and third (green) TPRs of Hsp90-binding three-TPR domains. The analysis was carried out on all TPR repeats from the Pfam database and separately on the three repeats of the Hsp90 binding proteins. The results for the six positions in the TPR sequence where the most significant differences were observed between the analysis of all TPR motifs data set and that performed using the Hsp90-binding TPR motifs are shown (P2, P5, P6, P7, P9, P23). (B) Amino acid sequence of the consensus TPR repeat CTPR3 (Main et al., 2003bGo) and schematic of the secondary structure of a TPR repeat. Sequences of the three TPR repeats, numbered 1, 2, 3, of the designed Hsp90-binding TPR, CTPR390, where positions that are conserved in Hsp90 binding proteins and which were incorporated into the peptide-binding design are highlighted in dark blue and conserved positions that where already present in CTPR3 are highlighted in pale blue. The mutations incorporated to re-engineer the charge on the back/convex face are shown in red. The protein has an N-capping sequence, GNS, at the N-terminus and a solvating helix after the final TPR repeat AKAKQNLGNAKQKQG. (C) Ribbon diagram of modeled CTPR390 based on CTPR3 crystal structure. The side chains of the binding residues conserved in Hsp90-binding proteins are shown.

 
Figure 1A illustrates that there are certain positions where the preference for a specific amino acid is different in the Hsp90-binding TPRs, in comparison with all TPRs. Notably, the positions that we identified as a result of this statistical analysis of linear sequences fit well with the residues in HopTPR2A that were identified as essential for the interaction of this domain with the C-terminal peptide of Hsp90 in independent biochemical and structural studies (Scheufler et al., 2000Go). Moreover, these residues are on the front/concave surface of the TPR, which is where the peptide is seen to bind in the co-crystal structure of TPR2A of Hop with the C-terminal peptide of Hsp90.

Because the Hsp90-binding dataset contains a small number of sequences (nine), only positions where a substantial difference in the amino acid preference for Hsp90-binding TPRs versus all TPRs were included. These residues, which are clearly a common feature of only Hsp90-binding TPR proteins (Figure 1A and B), were introduced into CTPR3: in the first TPR repeat, K in position 5 and N in position 9; in the second TPR repeat N in position 6 and in the third TPR repeat K in position 2, R in position 6, R in position 7 and D in position 23.

In addition, there are a few positions where the significance of the statistical analysis is less clear. An additional mutation at position 2 of the second repeat was incorporated, where, considering the sum of the frequency of Ser and Thr, it is clear that a hydroxylamino acid is preferred over the acidic Glu that appears in the consensus sequence. Ser was chosen because it showed a higher frequency than Thr.

Natural TPR motifs have near-zero net charge; the average net charge for 6887 TPR motifs is +0.04 (T.J.Magliery and L.Regan, submitted). For example, the values for the PP5 and TPR2A domain of Hop, two natural TPR domains that interact with Hsp90, are +4 and +2, respectively. By contrast, CTPR3 has a net charge of –15, mostly because of its highly negatively charged back/convex face (Figure 2).

It is interesting that in spite of the dramatically skewed charge distribution in CTPR3, the protein adopts the typical TPR fold and it is in fact more stable than most average, ‘charge balanced’ natural three-TPR domains. The Tm of CTPR3 is 84°C, whereas the three-TPR domain of PP5 has a Tm of 44°C (C.Wilson and L.Regan, unpublished data) and TPR1 and TPR2A domains of Hop have Tms of 50 and 56°C, respectively (T.Kajander and L.Regan, unpublished data). It appears that other interactions that stabilize the TPR fold are more than sufficient to overcome the energetically unfavorable charge distribution. It also indicates that surface salt bridges do not play a major role in specifying the TPR fold.

Although CTPR3 is stable and well folded, our aim is to bind a negatively charged ligand (MEEVD-COO), and we therefore re-engineered the charge on the back/convex surface of the protein. An analysis of the distribution of charged amino acids on the back/convex surface of CTPR3, reveals several examples where a negatively charged residue (D or E) has the highest global propensity, but the residue with the second highest global propensity value, with a probability of occurrence close to the first one, is a positively charged residue (K), for example at positions 16, 19, 22 and 29.

In crystal structures of natural TPR domains we observed that there are salt bridges on the back/convex face, resulting from positive/negative charge complementarity at the i, i + 3, i + 4 positions on this solvent-exposed surface. This charge complementarity has been artificially removed in the pure consensus sequence. The back/convex face of CTPR3 was therefore redesigned, changing a negatively charged residue (E) for a positively charged residue (K) at position 19 of the three TPR repeats. A charge–charge network was thus introduced on to the back/convex face of the protein with alternatively a negative or a positive residue every three positions. (i.e. negative residues at positions 16 and 22 and a positive residue at position 19).

A statistical free energy analysis (Lockless and Ranganathan, 1999Go) of natural TPR proteins suggests that there are interactions to balance charge between positions 16 and 19 and between positions 26 and 29. Correlations are also seen between positions 19, 22 and 26 (T.J.Magliery and L.Regan, submitted).

An additional mutation was incorporated at position 18, changing D to Q, the residue with the third highest global propensity value after D and E, in the three TPR repeats. The redesigned protein, CTPR390, presents a net charge of +1 and an electrostatic potential distribution on the back/convex face comparable that of to natural TPR domains (Figure 2).

Structure

The gene encoding CTPR390 (CTPR3–Hsp90 binding) was synthesized and the protein expressed and purified. Purified CTPR390 is very soluble and monomeric in solution. It exhibits a typical {alpha}-helical CD spectrum, virtually identical with that of CTPR3, with an MRE at 222 nm of –22 460 deg cm2/dmol (Figure 3A). The 2D NMR 1H–15N HSQC spectrum of CTPR390 (Figure 3D) is consistent with a well-folded and structured protein, with 116 resolved cross peaks (out of a possible 125) and good peak dispersion, comparable to the spectrum of CTPR3.



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Fig. 3. Solution characterization of the CTPR390 protein. (A) Far-UV CD spectra of CTPR3 (solid line) and CTPR390 (dashed line). (B) Thermal denaturation of CTPR3 (solid line) and CTPR390 (dashed line) monitored at 222 nm. (C) Gu·HCl equilibrium denaturation data for CTPR3 (filled circles) and CTPR390 (open circles) followed by CD at 222 nm. Fraction unfolded data were fitted to a two-state model (solid lines). (D) 1H–15N HSQC NMR spectrum of CTPR390 recorded in 150 mM NaCl, 10% D2O, 50 mM phosphate, pH 6.3 buffer at 20°C.

 
Stability

The thermal denaturation curve monitored by measuring the ellipticity at 222 nm for CTPR390 shows a cooperative and reversible behaviour. The transition appears slightly less cooperative than that of CTPR3, but CTPR390 is more resistant to thermal denaturation than CTPR3; it is not completely denatured by 95°C (Figure 3B). The Gu·HCl equilibrium unfolding was measured by CD, monitoring the changes in the ellipticity at 222 nm at increasing concentration of Gu·HCl. CTPR390 shows a cooperative and reversible unfolding transition with a midpoint (D1/2) of 3.28 M Gu·HCl. The calculated stability ({Delta}GH2O) of CTPR390 in the absence of denaturant is –12 kcal/mol (Figure 3C). The thermodynamic stability of CTPR390 is slightly higher than that of CTPR3, which has a D1/2 of 3.05 M and {Delta}GH2O of –10 kcal/mol.

Ligand binding

The TPR2A domain of Hop interacts with the C-terminal tail of the chaperone Hsp90. TPR2A binds to a five amino acid peptide corresponding to this tail, with the same affinity as it binds to full-length Hsp90. We therefore tested the binding of the designed TPR proteins against synthetic peptides that incorporate this C-terminal sequence.

The binding of TPR constructs to the C-terminal peptide of Hsp90 was performed using SPR. A 24-mer peptide, corresponding to the C-terminal amino acids of Hsp90, was biotinylated at the N-terminus and immobilized on a neutravidin-coated sensor chip. Increasing concentrations of the test TPR were passed over the derivatized chip surfaces and response units were monitored. The equilibrium response levels (Req) were calculated (subtracting background binding to neutravidin chip alone) and plotted against the protein concentration (Figure 4A).

CTPR390 binds to the C-terminal peptide of Hsp90 in a concentration-dependent, saturable manner, indicative of a specific protein–peptide interaction. A dissociation constant (KD) of 200 µM was determined by fitting the equilibrium binding data to a simple 1:1 binding scheme. We assayed the parent protein, CTPR3, in exactly the same fashion. CTPR3 showed no detectable binding to the Hsp90 peptide, up to protein concentrations of 1 mM (Figure 4A).

The binding is specific

Natural TPR domains discriminate between similar ligands in a highly specific manner. For example, the TPR1 and TPR2A domains of the protein Hop bind specifically to the C-terminal peptides of Hsp70 and Hsp90, respectively. Approximately 10-fold weaker binding of TPR1 to Hsp90 and of TPR2A to Hsp70 was reported in a cross-partner binding assay by isothermal titration calorimetry (Scheufler et al., 2000Go) and we have obtained similar results by SPR (T.Kajander and L.Regan, unpublished data). Because the last four residues of the Hsp70 and Hsp90 C-terminal peptides are identical (EEVD), specificity is mainly achieved by hydrophobic contacts with residues N-terminal to this sequence. The conformation in which the EEVD is bound may also contribute to binding specificity.

We therefore compared the binding of CTPR390 to the C-terminal peptides of both Hsp90 and Hsp70. As shown in Figure 4B, CTPR390 specifically binds to Hsp90. No significant binding to Hsp70 C-terminal peptide is detected. This result is important, because although the affinity of CTPR390 for the Hsp90 peptide is ~40-fold weaker than that measured for natural TPR domains binding to the C-terminal peptide of Hsp90 [TPR2A KD {approx} 4.5 µM, mSTI1 KD {approx} 5 µM (Odunuga et al., 2003Go)], the specificity of the protein–peptide interaction and discrimination between similar peptide ligands is clearly reproduced.

Importance of the back/convex face electrostatic charge

The parent protein, CTPR3, has a highly negatively charged back/convex face (Figure 2), which we altered in the CTPR390 design. The effect on ligand binding of the surface charge on the back/convex face was examined with the protein CTPR390-B. CTPR390-B has the same mutations on the front/concave binding face as CTPR390, but the back/convex face maintains the negatively charged residues of CTPR3. CTPR390-B is helical and folded by both CD and 1H–15N HSQC NMR analysis. CTPR390-B shows no interaction with the C-terminal peptide of Hsp90, even up to 800 µM protein concentration (data not shown). This result demonstrates that the charge of the back/convex surface of the protein, although distal from the ligand-binding surface, can exert a significant effect on ligand binding.

Estimating the contribution of the charge on the back/convex face to binding affinity

To investigate the effect of overall protein charge on ligand binding, we performed continuum electrostatics calculations with the program DelPhi (Gilson and Honig, 1987Go). We calculated the electrostatic energies of binding (see Materials and methods) at concentrations of monovalent cations and anions equivalent to the range 0–300 mM salt (Table I, Figure 5) for CTPR390, which has a neutralized back/convex face and a net charge of +2, and CTPR390-B, which has a negatively charged back/convex face and net charge of –7. Models for TPR–peptide complexes were generated as described in the Materials and methods. No attempt was made to model short-range interactions, beyond avoidance of steric clashes, because one would expect all the close-range interactions to be identical in both complexes. The effects of direct short-range interactions will therefore be subtracted out and calculated differences between the two complexes will reflect the influence of the back/convex surface charge.


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Table I. Calculated estimates for electrostatic energies of binding for CTPR390 and CTPR390-B as a function of salt concentration (energies are given in kcal/mol)

 


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Fig. 5. Plot of the values of the calculated estimates for electrostatic energies of binding for CTPR390 and CTPR390-B as a function of salt concentration (energies are given in kcal/mol). Data points for CTPR390 are indicated with open circles connected with a dashed line and data points for CTPR390-B are indicated with filled circles connected with a solid line.

 
The results of these calculations indicate that in the absence of salt, the favorable electrostatic contribution to binding is greater for CTPR390 (neutral back face) than for CTPR390-B (negatively charged back face) by about 4 kcal/mol (Table I).

At low concentrations of salt, the electrostatic contribution to the binding energy of the CTPR390-B–peptide interaction increases. Presumably, to some extent the salt shields the repulsive electrostatic interactions between the negatively charged CTPR390-B and the negatively charged peptide.

As the concentration of salt increases, the electrostatic contributions to binding of both CTPR390 and CTPR390-B are diminished, until at high salt concentrations the energies converge and both continue to decrease as the salt concentration increases still further (Table I, Figure 5). The primary effect of high concentrations of salt is to shield the favorable protein–peptide electrostatic interaction.

In summary, the calculations demonstrate how unfavorable negative charge on the back face can decrease binding affinity, as observed experimentally. These results also suggest that it should be possible to enhance the affinity for a ligand by appropriately tunning the charge on the back/convex face.

Chemical shift perturbation mapping of the CTPR390–Hsp90 C-terminal peptide interaction

We used 1H–15N HSQC NMR chemical shift perturbation experiments to map the area of CTPR390 involved in interactions with the Hsp90 C-terminal peptide. First, the HSQC spectrum of CTPR390 alone was acquired, then aliquots of peptide were added, after each addition samples were equilibrated for 30 min and an HSQC spectrum was acquired. By monitoring changes in the chemical shifts of particular NH peaks in different regions of the HSQC spectrum, we were able to identify which backbone NH resonances change upon peptide binding (Figure 6A).



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Fig. 6. HSQC perturbation of CTPR390 with C-terminal Hsp90 peptide. (A) Close-up of an overlay 1H–15N HSQC NMR spectrum of 15N-CTPR390 in the presence (green, 2 equiv.; blue, 3 equiv.) and absence (magenta) of C-terminal peptide of Hsp90. Labels show the residue type and number for the cross peaks that changed on binding of the peptide. (B) Ribbon diagram of modeled CTPR390 based on CTPR3 crystal structure. The residues for which the 15NH signal was shifted are mapped on to the structure; their side chains are shown in orange.

 
Figure 6B shows the residues which exhibit chemical shift changes upon peptide binding mapped on to a model of the CTPR390 structure based on the CTPR3 crystal structure (Main et al., 2003bGo) and modeled using Swiss-Pdb Viewer (Guex and Peitsch, 1997Go).

All the residues that exhibit significant chemical shift perturbations on peptide binding (N42, N76, A43, Y54, Y66, K55, A77, A86, Y88) are located on the front/concave surface of the TPR structure. This is the surface where specific mutations were introduced with the aim of incorporating direct protein–peptide contacts. The positions of the shifted residues confirm that the CTPR390 is interacting with the peptide in the desired fashion.

None of the backbone NH resonances of the conserved ‘dicarboxylate clamp’ residues, which in the HopTPR2A–Hsp90 peptide complex bind the Asp side chain and C-terminal carboxyl groups (Scheufler et al., 2000Go), were seen to change upon peptide binding. We note that in the crystal structure of the HopTPR2A–Hsp90 complex none of the main chain NH protons of the ‘dicarboxylate clamp’ residues interact directly with the peptide (Scheufler et al., 2000Go). It may be that for these residues the NH signals detected in the HSQC are insensitive to side chain ligand binding. In this regard, when detecting changes in the main chain NH protons, the chemical shift experiment indicates a minimum number of interacting residues.

An alternative reason why no changes in the main chain NH chemical shifts are observed for the ‘dicarboxylate clamp’ residues is that they do not participate in peptide binding by CTPR390. We therefore investigated the interaction of the TPR with the C-terminal COO– group of the peptide in more detail as described below.

Significance of the carboxylate clamp in peptide binding to CTPR390

In HopTPR2A a ‘dicarboxylate clamp’ interacts with the side chain and C-terminal COO– of the terminal aspartate residue of the Hsp90. In TPR2A, the clamp is formed by residues K229, N233, N264, K301 and R305 (Scheufler et al., 2000Go). The equivalent residues are present in our designed protein CTPR390: K13, N17, N48, K78 and R82.

Both the side and main chain carboxylate groups of the C-terminal aspartate residue of Hsp90 are essential for tight binding to TPR2A. Mutating the C-terminal Asp to Ala decreases binding approximately 20-fold whereas amidation of the C-terminal COO– has an even greater effect, decreasing binding over 100-fold, to essentially unmeasurable (Brinker et al., 2002Go). Conversely, mutations of the clamp residues of TPR2A (Brinker et al., 2002Go), Cyp40 (Ward et al., 2002Go) and PP5 (Russell et al., 1999Go) have been shown to impair or severely reduce Hsp90-binding activity.

In view of these observations, we performed further studies to measure the interaction of CTPR390 with a C-terminally amidated Hsp90 test peptide. We observed that binding of CTPR390 to the amidated peptide, measured by SPR, was significantly decreased in comparison with binding to the peptide with a C-terminal carboxylate (Figure 7A). We also measured the binding of HopTPR2A domain to the C-terminally amidated peptide and found, as previously reported, that binding was greatly diminished (Figure 7B). These results suggest that there are interactions between the CTPR390 and the C-terminal carboxylate of the bound peptide. The diminished binding affinity of the amidated peptides in comparison with the peptides with C-terminal free carboxylate precludes analysis of the data in a more quantitative fashion.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The work presented here enhances our understanding of TPR–peptide recognition by delineating the minimal features that are necessary to incorporate specific ligand-binding activity on to a ‘bare’ TPR framework.

The functional-consensus strategy, although based on a limited number of sequences, successfully highlighted the particular residues involved in ligand binding. Thus, although there is only one co-crystal structure of a TPR protein bound to the C-terminal peptide of Hsp90 (TPR2A of HOP), the sequence analysis suggests that the other Hsp90-binding TPRs bind in the same fashion. This is important for functional design, because it implies a ‘direct readout’ in peptide recognition by three-TPR domains.

The designed protein, CTPR390, binds specifically to the C-terminal peptide of Hsp90, discriminating effectively against the related C-terminus of Hsp70. Thus, in addition to successfully binding the designed peptide, it distinguishes it from an extremely similar peptide. CTPR390 binds less tightly to the C-terminal peptide of Hsp90 than does the natural TPR2A domain of Hop. Future studies will delineate more exactly the details of peptide binding by CTPR390 that may be responsible for this compromise in affinity.

Our results demonstrate the usefulness of the TPR scaffold for functional design and hold promise for the creation of TPR domains with different binding specificities for potential biomedical or biotechnological applications.


    Acknowledgments
 
L.R. gratefully acknowledges the support of the NIH, GM49146. A.L.C. is the recipient of a postdoctoral fellowship from Spanish Ministry of Education, Culture and Sports. T.K. is supported by a postdoctoral fellowship from Helsingin Sanomat Centennial Foundation (Finland). We thank Thomas J.Magliery, Irina Pozdnyakova and Chris Wilson for careful reading and insightful comments on the manuscript.


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
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 References
 
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Received May 11, 2004; accepted May 17, 2004.

Edited by Alan Fersht