SUPPLEMENTARY MATERIAL

Transcription

1 SUPPLEMENTARY MATERIAL Section 1 Supp. Figure 1. Target-template alignments Alignments of each target sequence with their respective template sequence (PDB code) are shown in a) for attoc64-v TPR with protein phosphatase 5 (PP5, PDB:1A17), b) pptoc64-1 TPR with PP5 (1A17), c) pstoc64tpr (model I) with the Hop2a domain (1ELR) and in d) for pstoc64tpr (model II) with the Hop1 domain (1ELW). 1

2 a) attoc64-v MEASEVMKEKGNAAYKGKQWNKAVNFYTEAIKLNGANATYYCNRAAAFLEL 1A17 RDEPPADGALKRAEELKTQANDYFKAKDYENAIKFYSQAIELNPSNAIYYGNRSLAYLRT * * * * * * ** ** ** ** ** ** * * attoc64-v CCFQQAEQDCTKAMLIDKKNVKAYLRRGTARESLVRYKEAAADFRHALVLEPQNKTAKVA 1A17 ECYGYALGDATRAIELDKKYIKGYYRRAASNMALGKFRAALRDYETVVKVKPHDKDAKMK * * * * * *** * * ** * * * * * ** attoc64-v EKRLRKHI A17 YQECNKIVKQKAFERAIAGDEHKRSVVDSLDIESMTIEDEYSGPKL * b) pptoc IVSDGNSAAAELAKEKGNAAFKEKDYKKAVGFYTDAIRLNGNNATYYNNRAMAYLQL 1A17 RDEPPADGALKRAEELKTQANDYFKAKDYENAIKFYSQAIELNPSNAIYYGNRSLAYLRT ** ** * * ** *** * ** ** ** ** ** ** *** pptoc64-1 CSFSEAESDCTKALNLDKRSVKAYLRRGTAREFLGYYKEADEDFRQALIFEPTNKTASEA 1A17 ECYGYALGDATRAIELDKKYIKGYYRRAASNMALGKFRAALRDYETVVKVKPHDKDAKMK * * * * *** * * ** ** * * * * * pptoc64-1 LSRLKKLLYG A17 YQECNKIVKQKAFERAIAGDEHKRSVVDSLDIESMTIEDEYSGPKL * c) pstoc64 EQSAEISKEKGNQAYKDKQWQKAIGFYTEAIKLCGNNATYYSNRAQAYLELGSYLQAEED 1ELR GKQALKEKELGNDAYKKKDFDTALKHYDKAKELDPTNMTYITNQAAVYFEKGDYNKCREL * ** ** *** * * * * * * ** * * * * * * * pstoc64 CTTAISFDKKN VKAYFRRGTAREMLGYYKEAIDDFKYALVLE---PTNKRAAS 1ELR CEKAIEVGRENREDYRQIAKAYARIGNSYFKEEKYKDAIHFYNKSLAEHRTP----DVLK * ** * *** * * ** ** * pstoc64 SAERLRKLFQ ELR KCQQAEKILKEQERL * d) pstoc64 ---AEISKEKGNQAYKDKQWQKAIGFYTEAIKLCGNNATYYSNRAQAYLELGSYLQAEED 1ELW MEQVNELKEKGNKALSVGNIDDALQCYSEAIKLDPHNHVLYSNRSAAYAKKGDYQKAYED ***** * * * ***** * **** ** * * * ** pstoc64 CTTAISFDKKNVKAYFRRGTAREMLGYYKEAIDDFKYALVLEPTNKRAASSAERLRKLFQ 1ELW GCKTVDLKPDWGKGYSRKAAALEFLNRFEEAKRTYEEGLKHEANNPQLKEGLQNMEAR-- * * * * * * ** * * * highly conserved not conserved supp. Figure 1, Mirus et al.

3 Section 2 Analysis of the homology models and their MD simulation trajectories by principal components analysis To evaluate our homology models, we analyzed the average structures calculated from trajectories of the MD simulations. These averages are comparable to NMR ensembles, although the time scale accessible by NMR is orders of magnitudes larger than by MD simulation (Soares et al., 2004). Still, the ensembles generated by MD simulations are a powerful tool to investigate the dynamics of the modeled structures. To assess the quality of sampling of a local free-energy minimum for a molecule from the conformational space we applied the principal components analysis (PCA; Hess et al., 2000, 2002). First, the autocorrelation was computed for the principal components (PC) and the relaxation coefficients determined were averaged (column 7 in supp. Table 1) for the minimal number of PCs reflecting at least 80% of all fluctuations. Hess (2002) suggested that the simulation time should exceed ten times the correlation time (10*a) to have a high probability of sampling an entire free-energy minimum visited during the MD simulation. Accordingly, all simulations fulfill this criterion (supp. Table 1, column 2 vs. column 6). Secondly, the cosine content of the first principal component in dependence on the simulation length is almost zero at the end of the MD simulation (supp. Figure 2c). However, this is the most unreliable criterion (Hess, 2002). Therefore, we next analyzed the overlap of the covariance matrices in subintervals generated as described in materials and methods with the covariance matrix over the whole run. All models show an overlap larger than 0.8 after half of the simulation time (penultimate data point in supp. Figure 2d; supp. Table 1). Hess (2000, 2002) argues that an overlap of 0.8 and above indicates good sample space coverage. Finally, we inspected the root of the mean squared fluctuations (RMSF) of the C α atoms (supp. Figure 2e). All trajectories reach a ballistic behavior of the simulation time dependent RMSF. The maximal RMSF values are 2

4 listed in supp. Table 1. Hence, all models fulfill the criteria proposed by Hess (2000, 2002). Therefore, we conclude that we have sufficiently sampled conformations associated with the free energy minima that have been visited during the MD simulations and conclusions based on the simulations and the average structures are feasible. References Hess B. Similarities between principal components of protein dynamics and random diffusion. Phys Rev E 2000; 62: Hess B. Convergence of sampling in protein simulations. Phys Rev E. 2002; 65: Soares TA, Daura X, Oostenbrink C, Smith LJ, van Gunsteren WF. Validation of the GROMOS force-field parameter set 45Alpha3 against nuclear magnetic resonance data of hen egg lysozyme. J Biomol NMR 2004; 30: Supp. Table 1 Results of the principle components analysis of the MD simulations of the homology models Model time a avg. RMSD b aa in PCA c max RMSF max overlap e a (ns) f (ns) (Å) (Å) d pstoc64 (I) 60 ns ±0.1 pstoc64 (II) 50 ns ±0.01 attoc64-v 50 ns ±0.01 pptoc64 50 ns ±0.01 attoc64-v 80 ns ±0.02 (Hsp90) (1.73) g pstoc64 (II)(Hsp90) 80 ns 1.47 (1.47) g ±0.02 a the period of MD simulation performed for the indicated model. b average RMSD calculated excluding the first nanosecond (for the free 3-TPR domains) or the first 5 nanoseconds (for the complexes). c residues of the homology models that were taken into account for the PCA. d the averaged RMSF of the entire simulation time as determined by PCA. e The overlap of the principle components of the half trajectory with the entire trajectory was calculated according to Hess (2002). f calculated from the exponential decay of the autocorrelation of the principal components according to Hess (2002). g average RMSD of the peptide 3

5 Supp. Figure 2. Comparison of 3-TPR domain models and principal component analysis Data for model I of pstoc64tpr are shown in grey, for model II of pstoc64tpr are shown in blue, for the model of attoc64-v TPR in cyan and date of pptoc64-1 TPR in red. (a) Shown are the changes of the RMSD of the C α atoms of each model of pstoc64tpr with respect to the starting structure of each trajectory. The trajectory of model I of pstoc64tpr was constructed by concatenating the trajectories of three MD simulations (supp. Figure 2a). The 2 nd and 3 rd MD simulation represent continuations of the 1 st MD simulation at time 928 ps and 2034 ps, respectively. For model I of pstoc64tpr we observed a rise of the RMSD up to the 6 th ns of the first part of the tripartite MD simulation followed by a decline to a lower level. The second MD simulation (nanoseconds 20 to 40) also showed stronger fluctuations. However, the third MD simulation (nanoseconds 40 to 60) dropped very fast into a low equilibrium fluctuating around an RMSD of 2.2Å. In contrast, after a quick rise during the first 500ps model II remained on average at a RMSD of 2.0Å. (b) Root mean square fluctuation of the C α atoms of each model of pstoc64tpr is shown. Helices are indicated as boxes above the x-axis, loops as lines connecting these boxes. In (c) and (d) the results of the PCA are plotted. (c) shows the -dependent decay of cosine content of the first principal component and (d) the average overlap of the covariance matrices of each subinterval of the trajectories with the covariance matrix of the full trajectory (see materials and methods). In (e) the root means square fluctuation (RMSF) is plotted as a function of the length of the subinterval of the trajectories. 4

6 A) RMSD [nm] B) RMSF [nm] Model I Model II time [ns] C) cosinus content D) overlap amino acid E) RMSF [nm] interval [1/a] supp. Figure 2, Mirus et al.

7 Section 3 Analysis of the peptide-tpr complex by principal component analysis The average RMSD of the liganded 3-TPR domain of attoc64-v (supp. Figure 3a), pstoc64 (not shown) and of the bound octapeptide was analyzed (supp. Fig. 3b, black). In order to explore the movements of the ligand relative to the receptor within the binding groove we superimposed the complex by the C α atoms of the core residues (supp. Fig. 3d) of the 3- TPR domain only. This analysis shows that the ligand moves quite strongly within the binding groove (RMSD > 0.6 nm in parts, supp. Figure 3b, grey). The analyses of the RMSD distribution of both, the Arabidopsis and the pea systems show that the internal motions of the 3-TPR domain are more restricted when the peptide is bound (supp. Fig. 3c, top). However, when the octapeptide is complexed to the 3-TPR domain of Toc64 from pea the restriction of the internal movement of the structure is not as pronounced as obtained for attoc64-v TPR (supp. Fig. 3c, top). The distribution of the RMSD of the octapeptide suggests the existence of at least two states for both, the Arabidopsis and the pea model (supp. Fig. 3c, bottom). In the Arabidopsis model the peptide resides dominantly in one state (supp. Fig. 3c, bottom). A quadruple Gaussian fit revealed that the area below the two most prominent peaks in the pea model is quite similar. Performing a PCA of the 3-TPR domains of the complexed systems we obtained a similar quality of the sampling as for the free 3-TPR domain (supp. Figure 2, 4, supp. Table 1). Hence, we conclude that at least for the two 3-TPR domains of pstoc64 and attoc64 the sampling is sufficient for further analysis of the complexes. We next analyzed the conformations taken up by the peptide. As a reference the starting structure of the ligand of attoc64-v TPR was used to superimpose the trajectories of both ligands from the MD simulations of attoc64-v TPR and pstoc64tpr by the coordinates of their C α atoms. The trajectories were concatenated leaving out approximately the first 6 ns each to remove equilibration effects - and a RMSD matrix was calculated. The RMSD matrix 5

8 was constructed by calculating pairwise RMSDs of the structures within the trajectory. With this matrix we performed a cluster analysis with GROMACS g_cluster tool choosing gromos clustering method (Daura et al., 1999) with a cutoff of 0.2 nm including every 10 th frame of the trajectory. The majority of the conformations of both 3-TPR domains ligands was assigned to a single cluster (supp. Figure 5a, b). It is obvious, that a significant portion of conformations of the ligand of pstoc64tpr is different from the Arabidopsis system (supp. Fig. 5a, b). References Daura X, Gademann K, Jaun B, Seebach D, van Gunsteren WF, Mark AE. Peptide folding: When simulation meets experiment. Angew. Chem. Int. Ed. 1999; 38:

9 Supp. Figure 3. Peptide binding by the 3-TPR domain of attoc64-v and pstoc64 (a) The RMSD for the MD simulations of attoc64-v with (grey) and without (black) peptide are shown. (b) The RMSD of the peptide was determined by superimposing it onto its starting structure (black) or relative to the C α atoms of the 3-TPR domain (grey). (c) The RMSD distribution in relation to the average RMSD calculated from the MD trajectory for pstoc64tpr without (top, grey circle) and with bound peptide (top, grey triangle), for attoc64-v TPR without (middle, black circle) and with bound peptide (middle, black triangle), as well as for the peptides complexed to attoc64-v TPR (bottom, black square) or pstoc64tpr (bottom, grey square) is shown. The bin width for all was set to (d) RMSD comparison between entire 3-TPR domain, the 3-TPR core and the flexible regions of attoc64-v TPR. The RMSD (C α atoms) of residues , , , and (green, representing the stable core of the domain), of residues , , , , and (red, residues not in the core region), and of all residues within the TPR core domain ( , grey) is shown for comparison. 7

10 C) A) RMSD Cα [nm] B) percent percent time [nsec] 0.30 RMSD-RMSDavg D) RMSD Cα [nm] time [nsec] supp. Figure 3, Mirus et al. 80

11 Supp. Figure 4. Principal components analysis of the complexed 3-TPR domain models The overlap of the sampling of each subinterval with the sampling of the entire run for attoc64-v (black) and pstoc64 (grey) complexed with the peptide is given as described in supp. Figure 2. 8

12 RMSF [nm] overlap interval [units of a] supp. Figure 4, Mirus et al.

13 Supp. Figure 5. Analysis of ligand behavior from MD simulations For (a) the trajectories of the C α atoms of each ligand (from attoc64-v TPR and pstoc64tpr) were concatenated (each excluding roughly the first 6 ns) and a RMSD matrix calculated (upper half of matrix). The lower half of the matrix displays the result of clustering the ligands by the RMSD matrix with the GROMACS tool g_cluster using a cutoff of 0.2 nm for the gromos method (Daura et al. 1999). In (b) we have shown how many of the stored structures of each 3-TPR domain s ligand were assigned to the clusters determined in (a). c) From the eigenvectors of the ligands (covariance matrices of C α atoms) we computed a matrix of inner products. (d) shows for each eigenvector its eigenvalue normalized to the sum of all eigenvalues. The smaller graph in (b) and (d) holds the same data, but the y-axis is scaled logarithmically. 9

14 A) pstpr B) attpr time [ps] attpr C) pstpr time [ps] D) 24 pstpr - ligand attpr - ligand supp. Figure 5, Mirus et al.

15 Section 4 Mapping the modeled interaction between peptide and 3-TPR domain and motions within the 3-TPR domains with and without ligand Analysis of the MD simulations with respect to the dynamics of the H-bond network between 3-TPR domain and octapeptide (supp. Figure 6) is described in the manuscript. For the analysis of the motion within the simulated 3-TPR domains we used the PCA. To compare the MD simulations of the 3-TPR domain of attoc64-v and pstoc64 (with and without ligand) and of pptoc64 we have calculated the inner products of the first few eigenvectors. The different types of motions held within eigenvectors 1 to 3 are very similar for all simulated homology models of the unliganded 3-TPR domain (supp. Figure 9a). On the other hand, eigenvector 4 matches best between attoc64-v and pptoc64-1 only. To identify motions covered by each eigenvector, the direction and strength of the motion was determined and visualized by cones originating from the C α atoms of the average structure (Tai et al., 2001) representing both by their direction and length, respectively (supp. Figure 9c). For eigenvector 1 (supp. Figure 9c, left) it is clearly visible that helix b of TPR motif II remains stationary, while the N terminal and C terminal attached parts move like a metronome or pendulum (depending on the point of view). In this context the short loops of helix IIb act as hinges. The 2 nd eigenvector (supp. Figure 9c, middle) holds an opening/closing motion of the peptide binding site with helix IIb acting as the rotational axis. In eigenvector 3 (supp. Figure 9c, right), the TPR motifs fan out at the side of the intrarepeat loops. Supp. Figure 3 (a, c) shows that the presence of the octapeptide within the binding groove of the 3-TPR domain restricts the flexibility of this domain, as can be inferred from the reduced RMSD variations. But which internal motions are affected, and which motions are dominant in the complexes? Pairwise inner-products of the first four eigenvectors of the free 3-TPR domain and its liganded form show that the motions previously observed in the unliganded 3- TPR domain have changed their ranking for both attoc64-v and pstoc64 (supp. Fig. 9a). It 10

16 becomes obvious that the movements of loop 4 (PC 2, 3) and of the solvation helix (PC 1) are more prevalent when the 3-TPR domain of attoc64-v is complexed with a peptide (supp. Fig. 9c). The same holds true for loop 2 and the solvation helix when pstoc64tpr is complexed (not shown). Analyzing the difference of the RMSF of the unliganded and liganded 3-TPR domain (Fig. 3A) reveals that the two loops indeed show stronger movement, but not so the solvation helix. Taken together the results suggest that, with exception of the two mentioned loops (2 and 4), the peptide reduces the freedom of motion of the 3-TPR domain. In contrast to the 3-TPR domains (supp. Fig. 9a), about 80% of the motions of the ligands are represented by the first eigenvector (supp. Fig. 5c, d). However, the inner product of the first eigenvectors of the covariance matrices of the ligands from the MD simulations of the 3-TPRdomain-peptide complexes is only This shows that the dominant motion is similar, and it also reflects the distinctive properties of both 3-TPR domains. References Tai K, Shen T, Börjesson U, Philippopoulos M, McCammon JA. Analysis of a 10-ns molecular dynamics simulation of mouse acetylcholinesterase. Biophys J 2001; 81:

17 Supp. Table 2 Assignment of residues to indices in Supp. Fig. 6 attoc64-v pstoc64 res res # index res res # index LYS LYS ASN ASN TYR TYR LYS LYS GLY LYS LYS THR THR SER ASN ASN GLU GLN ASN GLU LYS ASN ARG LYS THR ARG GLU THR SER GLU TYR MET ASN ASN LYS LYS THR ARG VAL SER ALA SER LYS ARG ARG LEU LEU LYS ARG LYS HIS

18 Supp. Table 3 Intramolecular H-bonds of the 3-TPR domain of attoc64-v TPR were determined with the g_hbond tool of GROMACS v as described in materials and methods, but without solvent insertion. The atom name, its residue and sequence position are given for the donor and acceptor atom of an H-bond. The last column contains the number of frames of the trajectory, in which the H-bond was present. Only H-bonds, which were detected in at least 20% of the frames of the trajectory (minus the first 5 ns as equilibration time), are listed. Donor Acceptor number atom residue atom residue of frames OG SER 488 ND2 ASN OG SER 488 OH TYR NZ LYS 492 OE1 GLU NZ LYS 492 OE2 GLU ND2 ASN 496 OE1 GLU ND2 ASN 496 OE2 GLU OH TYR 511 OD1 ASN OH TYR 511 ND2 ASN ND2 ASN 518 ND2 ASN ND2 ASN 521 OD1 ASN OH TYR 524 OE1 GLU OH TYR 524 OE2 GLU NE ARG 528 OG1 THR NE ARG 528 OD1 ASP NE ARG 528 OD2 ASP NH1 ARG 528 OG1 THR NH2 ARG 528 OD1 ASP NH2 ARG 528 OD2 ASP NE2 GLN 539 OE1 GLU NE2 GLN 539 OE2 GLU OG1 THR 546 OE1 GLU OG1 THR 546 OE2 GLU NZ LYS 547 OD1 ASP NZ LYS 547 OD2 ASP NE ARG 562 OD1 ASP NE ARG 562 OD2 ASP NH1 ARG 562 OG1 THR NH2 ARG 562 OD1 ASP NH2 ARG 562 OD2 ASP NE ARG 566 OE1 GLU NE ARG 566 OE2 GLU NH1 ARG 566 OD1 ASP NH1 ARG 566 OD2 ASP NH2 ARG 566 OE1 GLU ND2 ASN 589 OE1 GLU ND2 ASN 589 OE2 GLU

19 Supp. Table 4 Intramolecular H-bonds of the 3-TPR domain of attoc64-v TPR in its Hsp90-bound state were determined with the g_hbond tool of GROMACS v as described in materials and methods, but without solvent insertion. The atom name, its residue and sequence position are given for the donor and acceptor atom of an H-bond. The last column contains the number of frames of the trajectory, in which the H-bond was present. Only H-bonds, which were detected in at least 20% of the frames of the trajectory (minus the first 5 ns as equilibration time), are listed. Donor Acceptor number atom residue atom residue of frames OG SER 488 ND2 ASN OG SER 488 OH TYR NZ LYS 492 OE1 GLU NZ LYS 492 OE2 GLU ND2 ASN 496 OE2 GLU OH TYR 511 OD1 ASN OG1 THR 512 NH2 ARG OH TYR 524 OE1 GLU OH TYR 524 OE2 GLU ND2 ASN 527 OD1 ASN NE ARG 528 OG1 THR NE ARG 528 OD1 ASP NE ARG 528 OD2 ASP NH1 ARG 528 OG1 THR NH1 ARG 528 OD1 ASP NH1 ARG 528 OD2 ASP NH2 ARG 528 OD1 ASP NH2 ARG 528 OD2 ASP NZ LYS 547 OD1 ASP NZ LYS 547 OD2 ASP NE ARG 562 OD1 ASP NE ARG 562 OD2 ASP NH1 ARG 562 OG1 THR NH2 ARG 562 OD1 ASP NH2 ARG 562 OD2 ASP NE ARG 566 OE1 GLU NE ARG 566 OE2 GLU NH1 ARG 566 OD1 ASP NH1 ARG 566 OD2 ASP NH2 ARG 566 OE1 GLU NH2 ARG 566 OE2 GLU

20 Supp. Table 5 Intramolecular H-bonds of the 3-TPR domain of pstoc64tpr were determined with the g_hbond tool of GROMACS v as described in materials and methods, but without solvent insertion. The atom name, its residue and sequence position are given for the donor and acceptor atom of an H-bond. The last column contains the number of frames of the trajectory, in which the H-bond was present. Only H-bonds, which were detected in at least 20% of the frames of the trajectory (minus the first 5 ns as equilibration time), are listed. Donor Acceptor number atom residue atom residue of frames OG SER 480 OE1 GLU OH TYR 488 NE2 GLN OH TYR 488 OE1 GLU OH TYR 488 OE2 GLU OH TYR 500 OD1 ASN OH TYR 500 ND2 ASN OG1 THR 501 NH1 ARG ND2 ASN 509 OH TYR ND2 ASN 510 OH TYR OH TYR 513 OE1 GLU OH TYR 513 OE2 GLU OH TYR 514 OD1 ASN OG SER 515 NH1 ARG ND2 ASN 516 OE1 GLN NE ARG 517 OD1 ASP NE ARG 517 OD2 ASP NH2 ARG 517 OD1 ASP NH2 ARG 517 OD2 ASP NE ARG 551 OG1 THR NE ARG 551 OD1 ASP NE ARG 551 OD2 ASP NH1 ARG 551 OG1 THR NH2 ARG 551 OD1 ASP NH2 ARG 551 OD2 ASP NE ARG 555 OE1 GLU NE ARG 555 OE2 GLU OG1 THR 577 OE1 GLU OG1 THR 577 OE2 GLU OG SER 584 OE1 GLU OG SER 584 OE2 GLU

21 Supp. Table 6 Intramolecular H-bonds of the 3-TPR domain of pstoc64tpr in its Hsp90-bound state were determined with the g_hbond tool of GROMACS v as described in materials and methods, but without solvent insertion. The atom name, its residue and sequence position are given for the donor and acceptor atom of an H-bond. The last column contains the number of frames of the trajectory, in which the H-bond was present. Only H-bonds, which were detected in at least 20% of the frames of the trajectory (minus the first 5 ns as equilibration time), are listed. Donor Acceptor number atom residue atom residue of frames OG SER 480 OE1 GLU OG SER 480 OE2 GLU NZ LYS 481 OE1 GLU OH TYR 488 NE2 GLN OH TYR 500 OD1 ASN ND2 ASN 510 OG1 THR OH TYR 513 OE1 GLU OH TYR 513 OE2 GLU OG SER 515 NH1 ARG ND2 ASN 516 OD1 ASN NE ARG 517 OD1 ASP NE ARG 517 OD2 ASP NH1 ARG 517 OG1 THR NH2 ARG 517 OD1 ASP NH2 ARG 517 OD2 ASP OG1 THR 535 OE1 GLU OG1 THR 535 OE2 GLU NE ARG 550 OE1 GLN NE ARG 551 OD1 ASP NE ARG 551 OD2 ASP NH1 ARG 551 OG1 THR NH2 ARG 551 OD1 ASP NH2 ARG 551 OD2 ASP OG1 THR 577 OE1 GLU OG1 THR 577 OE2 GLU

22 Supp. Table 7 Non-bonded Coulomb (cl) and Lenard-Jones (lj) energies between attoc64-v TPR residues and ligand in [kj*mol -1 ] rec res num rec res res-lig[cl] res-lig[lj] res-sol[cl] res-sol[lj] 492 Lys e Asn e Tyr e Lys e Lys e Trp e Tyr e Thr e Cys e Asn e Ala e Leu e Glu e Cys e Phe e Asn e Lys e Leu e Arg e Thr e Glu e Ser e Tyr e Phe e Asn e Thr e Val e Ala e Arg e Leu e-4 17

23 Supp. Table 8 Non-bonded Coulomb (cl) and Lenard-Jones (lj) energies between pstoc64tpr residues and ligand in [kj*mol -1 ] rec res num rec res res-lig[cl] res-lig[lj] res-sol[cl] res-sol[lj] 481 Lys e Asn e Tyr e Lys e Lys e Trp e Tyr e Thr e Ser e Asn e Gln e Ala e Leu e Glu e Tyr e Asn e Lys e Phe e Arg e Thr e Glu e Met e Tyr e Phe e Asn e Lys e Arg e Ser e Ser e Arg e Leu e Leu e Phe e-4 18

24 Supp. Figure 6. H-bond network between Toc64 3-TPR domain and its ligand (a) Shown are H-bonds observed during the MD simulations of the 3-TPR domain-peptide complex of attoc64-v and pstoc64, respectively, with the C-terminal 8-mer of Hsp90. These plots were constructed by joining the H-bond data (calculated by the GROMACS tool g_hbond (v3.1.4)) for all donor/acceptor atoms of each residue of the 3-TPR domain. Red marks a direct H-bond, blue stands for a water-mediated H-bond and yellow denotes a residue, which forms at the same time a direct and a water-mediated H-bond. Supp. Table 2 matches residues to the indices along the y-axis. (b, c) The minimal distance between an atom of the indicated amino acid and any atom of the peptide was calculated for each frame for the TPR of attoc64 (b) or pstoc64 (c). Shown is the percentage of the frames in which a distance between the two molecules is found which is below the indicated distance cut off. The legend is indicated on the right. 19

25 A) Hydrogen Bond Index pstoc64 attoc64-v time [ns] Hydrogen Bonds none present H20 inserted present&h20 inserted supp. Figure 6, Mirus et al.

26 Supp. Figure 7. Average helix crossangles Helix crossangles were calculated with YASARA for neighboring helices within each 3-TPR domain of attoc64-v and pstoc64 for each frame of the respective MD simulation. The average crossangle of each helix pair is shown with error bars representing the standard deviation. The free 3-TPR domains are compared with their liganded form bound to an octapeptide corresponding to the C-terminus of Hsp90. 20

27 supp. Figure 7, Mirus et al.

28 Supp. Figure 8. Contact maps Mean smallest pairwise residue distances were calculated with the GROMACS tool g_mdmat for the trajectories of the MD simulations of attoc64-v TPR and pstoc64tpr. The distance cutoff was set to 0.5 nm. The residue indices on the x and y axis map to residues (attoc64-v) and (pstoc64). In a) to c) half matrices are shown for a) attoc64-v TPR and b) pstoc64tpr. White marks a distance >0.5nm in a) and b). In c) the difference half matrices for attoc64-v TPR (upper left) and pstoc64tpr in the [free] minus [Hsp90] bound state are shown. Hence, positive values reflect distances between amino acids, which are longer in the non-liganded 3-TPR domain. White either means that the mean smallest distance is the same in the free and the Hsp90-bound state or that the distance in a) and b) was beyond the cutoff. 21

29 Mean smallest distance Mean smallest distance a) b) c) attoc64-v TPR attoc64-v TPR (Hsp90) pstoc64tpr pstoc64tpr (Hsp90) Residue Index Residue Index Residue Index Residue Index 0 Distance (nm) 0.5 supp. Figure 8, Mirus et al. Mean smallest distance attoc64-v [free]-[hsp90] pstoc64 [free]-[hsp90] Residue Index attoc64-v pstoc Distance (nm) Residue Index

30 Supp. Figure 9. Motions within the simulated 3-TPR domains (a) The inner products of the first four eigenvectors of the MD simulations of the 3-TPR domain of attoc64-v (free) and attoc64-v (liganded), pstoc64 (1ELW) and pptoc64-1, respectively (left); of pstoc64 (1ELW) and pptoc64-1, and pstoc64 (liganded), respectively (right top); and of attoc64-v (liganded) and pstoc64 (liganded) (right bottom) as a measure of similarity of the eigenvectors were calculated. The graphs show the fraction of the sum of the eigenvalues that is contained within the respective eigenvector. (b, c) The motions of the free (b) and liganded (c) 3-TPR domain of attoc64-v within eigenvector 1, 2 and 3 is visualized by cones originating from the C α atoms of the average structure representing both by their direction and length, respectively. 22

31 1.0 pstoc eigenvalue [fraction] pptoc eigenvalue [fraction] attoc64-v (Hsp90) pptoc pstoc attoc64-v (Hsp90) attoc64-v B) EV1 EV2 attoc64-v (Hsp90) C) pstoc64 (Hsp90) 1 pstoc64 (Hsp90) attoc64-v eigenvalue [fraction] A) 0.3 supp. Figure 9, Mirus et al. EV3

32 Section 6 Analysis of the amino acid conservation within the 3-TPR domain of the Toc64 family Supp. Figure 10. Amino acid conservation in the 3-TPR domain (a) Shown is a multiple alignment of the available Toc64 TPR domain sequences. The upper block contains all sequences of the plastidic sequences, the block at the bottom all sequences of the mitochondrial sequences. Red ellipses mark positions with an amino acid composition that differs between the two blocks. The orange ellipse marks a position, which shows a different amino acid composition in both blocks, but is involved in chaperone binding. Blue ellipses mark residues, which are conserved in both blocks and are involved in chaperone binding. Cyan ellipses mark positions which are conserved in both blocks, but are not involved in chaperone binding. 23

33 Mitochondria Plastids Atha (424) Atha (718) Osat (480) Ppat (584) Ppat (585) Psat Otau Oluc Ptric (921) Ptric (734) Ptric (EST) Apub Cric Lesc Mdom Sbic (859) Taes (671) Vvin EESAEIAKEKGNQAFKEKLWQKAIGLYSEAIKLSDNNATYYSNRAAAYLELGGFLQAEEDCTKAITLDKKNVKAYLRRGTAREMLGDCKGAI EDFRYALVLEPNNKRASLSAERLRKFQ- EESAEIAKEKGNQAFKEKLWQKAIGLYSEAIKLSDNNATYYSNRAAAYLELGGFLQAEEDCTKAITLDKKNVKAYLRRGTAREMLGDCKGAI EDFRYALVLEPNNKRASLSAERLRKFQ- EEAAEAAKEKGNIAFKEKQWQKAINFYTEAIKLNNKVATYYSNRAAAFLELASYRQAEADCTSAIDIDPKIVKAYLRRGTAREMLGYYKEAV DDFSHALVLEPMNKTAGVAINRLKKLFP SAAAELAKEKGNAAFKEKDYKKAVGFYTDAIRLNGNNATYYNNRAMAYLQLCSFSEAESDCTKALNLDKRSVKAYLRRGTAREFLGYYKEAD EDFRQALIFEPTNKTASEALSRLKKLLY AEAAEMAKEKGNASFKEKDYKKAISHYTDAIRMDENNATFYNNRAMAYLQLCSFQEAEADCTKALGLDKKSVKAYLRRGTAREFLGYYKEAN DDFRQAQILEPTNKTASEALARLKKLLI EQSAEISKEKGNQAYKDKQWQKAIGFYTEAIKLCGNNATYYSNRAQAYLELGSYLQAEEDCTTAISFDKKNVKAYFRRGTAREMLGYYKEAI DDFKYALVLEPTNKRAASSAERLRKLFQ REPGDAEKAKGNEALKKGKYQDAIEYYGVAIGKNPKNPVYVANRAMAHLKLGNYELCEDDCTTAIKLDRKYTKAYLRRATARSVGGNYLEAL MDFEEALRLEPNNSDAKREVNRMKKIIG EAPGESEKTKGNEALKQGKYQDAIEYYSVAIGKNPKSKIFVANRAMAHLKLGNYQLAEDDCTEAIKLDARYVKAYLRRAAARSVAGNYLEAL MDYEEALRFEPNNSDAKREVYRMKKIIG ENSAEMAKEKGNQAFKEQQWQKAISYYNEAIKLNDKNATYYSNRAAAYLELGSFQHAEADCSNAINLDKKNVKAYLRRGTAREMLGYYKDAI EDFKYALVLEPTNKRASLSAERLQKVFA ENSAEMAKEKGNQAFKEKQWKKAISYYNEAIKLNDKNATYYSNRAAAYLELGSFHQAEADCSKAINLDKKNVKAYLRRGTAREMLGYYKDAI EDFKYALVLEPTNKRASLSAERLRKVFP ENSAEMAKEKGNQAFKEQQWQKAISYYNEAIKLNDKNATYYSNRAAAYLELGSFQHAEADCSNAINLDKKNVKAYLRRGTAREMLGYYKDAI EDFKYALVLEPTNKRASLSAE EESAEIAKEKGNTAFKEKKMQKAIGFYTEAIKLNSDNATYYNNRAAAYLDVGSFLQAEADCSTAISLDKKNVKAYLRRGTAREMLGYYKEAI EDFRHALVLEPTNKRAALSADRLKKLFQ PEAAEAAKEKGNTAFKNKDFKTAIDYYSEAINCDGKNATYYNNRAAAYLAMCSFHQAEADCTTAIELDRKNVKSYLRRGTAREFLGFYKESD EDFGQALILEPTYNTAGLGVKRMGTLLD ETSAEMAKEKGNQAFKEKQWQRAIGFYTEAIKLNGNSATYFSNRAAAHLEMRNFLQAEADSSKAIDLDKKNVKAYLRRGTAREM EQSAEIAKEKGNQAYKDREWQKAIGFYSKXIKLXGNNATYYSNRAQAYLEVGSFIQAEADCTKAINLDKKNVKAYFRRGTAREMLGYYKEAI EDFRHTLVLEPTNKRAAVAAEKLRKLFL EEAAEAAKEKGNAAFKEKQWQKAVNFYTEAIKLNGKVATYYSNRAAAFLELTSYRQAEADCTSAIDLDPKSVKAYLRRGTAREMLGYYKDAV DDFNHALVLEPM EEAAEAAKEKGNSAFKEKQWQKAINLYTEAIKLNGKVATYYSNRAAAFLELANYRQAETDCTSAIDIDPKIVKAYLRRGTAREMLGYYKDAV DEFSHALVLEPMNKTAGVAINRLEKLFP ETSAEIAKEKGNQAFKDKQWQKAVGFYTEAIKLSGNNATYYSNRAAAYLEMGSFLQAEADCTKAINLDKKNVKAYLRRGTAREMLGYYKDAI EDFRYALVLEPTNKRASLSADRLKKLFQ Atha (504) MEASEVMKEKGNAAYKGKQWNKAVNFYTEAIKLNGANATYYCNRAAAFLELCCFQQAEQDCTKAMLIDKKNVKAYLRRGTARESLVRYKEAA ADFRHALVLEPQNKTAKVAEKRLRKHI- Atha (468) MEASEVMKEKGNAAYKGKQWNKAVNFYTEAIKLNGANATYYCNRAAAFLELCCFQQAEQDCTKAMLIDKKNVKAYLRRGTARESLVRYKEAAAGYWSVTLWLIISADFRHALVLEPQNKTAKVAEKRLRKHI- Osat (856) FGAAELLKEKGNSAFKGRKWSKAVEFYSDAIKLNGTNATYYSNRAAAYLELGRYKQAEADCEQALLLDKKNVKAYLRRGIAREAVLNHQEAL QDIRHALALEPQNKAGLLAERRLQKKLR Ptric (801) FDASELLKEKGNAAYKGKQWNKAVNYYSEAIKLNGKNATYYSNRAAAYLQLGCFQKAEEDCNMAISLDKKNVKAYLRRGTARESLLFYKDAA QDFKHALVLEPQNKVARHAEKRLRKLMS Acep MDVSELLKEKGNAAFKGKQWNKAVTLYSEAIKQNDSCATYYCNRAAAYLELGCFQQAETDCNQAISIDKKNVKAYLRRRTAREMLLC Ccle IDASELLKEKGNAAFKGKQWNKAVNYYSEAIKLNGTSATYYSNRAAAYLELGCFQQAEEDCSKTISLDKKNVKAYLRRGTAREALLYYNEA Hvul VDASELLKEKGNNSFKRKQWSKAIEFYSGAIKLNETNATYYCNRAAAYLELGRFKQAEADCDQALLLDKKNVKAYLRRGTAKESCMNYQEAL QDFRHALALEPQNKTALAAERRLQKHLR Pvul IESSDLLKEKGNAAFKGRLWNKAVNYYSEAINLNGTNATYFSNRAAAYLELGCFQEAEEDCNKAILHDKKNVKAYLRRGTARELLLRYEEAL KDFQHALVLEPQNKTASLAEKR Soff Sbic (842) VNASELLKEKGNSAFKRRQWSKAIEFYSEAISLSDSNATYYCNRAAAYLELGRLKQAEADCDRALLLDRKNVKAYLRRGCAREVTLNYKEAL QDFRHALALEPQNKTALAAERRLQKLLK KEKGNSAFKRRPWSKAIEFYSEAISLSDTNATYYCNRAAAYLELGRLKQVEGDCDRALLLDRKNVKAYLRRGCAREVTLNYKEAL QDFRHALALEPQNKTALAAERRLQKLLK Taes (188) VDTSELLKEKGNNSFKRKQWSKAIEFYSGAIKLNDTNATYYCNRAAAYLELGRFKQAEADCDQALLLDKKNVKAYLRRGTAKESVLNYQEAL QDFRHALALEPQNRTALAAEKRLQKHLR Zmay VNASELLKEKGNSAFKRRQWIKAIEFYSEAISLSDTNATYYCNRAAAYLELGRFKQAEADCDRALLLDRKNVKAYLRRGFAREVTLNYKEAL QDFRHALALEPQNKTALAAERRLQKLLK Ptrif IDASELLKEKGNAAFKGKQWNKAVNYYSEAIKLNGTSATYYSNRAAAYLELGCFQQAEEDCSKTISLDKKNVKAYLRRGTAREALLCYNEAL QDFKHAMVLEPQNKAANLAEKRLRKLIG Ptae IKAADISKEKGNVAFKGKQWHKAVNFYSEAIKLNDKNATYYSNRAAACLELGRFQQAEEDCSKAISIDKKNVKAYLRRGTARENLCYYTEAM EDFRYALVLEPTNKAASLAINRLKKLVD supp. Figure 10, Mirus et al.

34 Section 7 Checking the homology models References Lovell SC, Davis IW, Arendall WB 3rd, de Bakker PI, Word JM, Prisant MG, Richardson JS, Richardson DC (2003) Structure validation by Calpha geometry: phi,psi and Cbeta deviation. Proteins 50: Bowie JU, Lüthy R, Eisenberg D (1991) A method to identify protein sequences that fold into a known three-dimensional structure. Science 253: Lüthy R, Bowie JU, Eisenberg D (1992) Assessment of protein models with threedimensional profiles. Nature 356:83-85 Supp. Figure 11 Ramachandran plots Ramachandran plots were made by the Rampage server (Lovell et al., 2003; and are shown for the homology models of a) attoc64-v TPR, b) pstoc64tpr and c) pptoc64-1 TPR. 24

35 180 a) 180 b) ψ 0 ψ φ ψ c) φ General/Pre-Pro/Proline Favoured Glycine Favoured General/Pre-Pro/Proline Allowed Glycine Allowed Number of residues in favoured region (~98.0% expected) Number of residues in allowed region (~2.0% expected) Number of residues in outlier region a) attoc64-v TPR b) pstoc64tpr (model II) 116 (99.1%) 111 (96.5%) 1 (0.9%) 4 (3.5%) 0 (0.0%) 0 (0.0%) φ c) pptoc64-1tpr 124 (99.2%) 1 (0.8%) 0 (0.0%) supp. Figure 11, Mirus et al.

36 Supp. Figure 12 Model validation The Verify3D server (Bowie et al, 1991; Lüthy et al. 1992; was used to validate the homology models. The plots produced by the Verify3D server are shown for a) attoc64-v TPR, b) pstoc64tpr and c) pptoc64-1 TPR. 25

37 a) b) c) supp. Figure 12, Mirus et al.