Mip^77–213 was composed of the C-terminal FKBD and N-terminal amino acids that formed part of the α-helix connecting the two domains in full-length Mip. Statistics for the structure calculation are listed in table 1. The residues N-78 to N-95 of the shortened mutant formed a free-standing α-helix as also observed for the dimer (Figure 2). The C-terminal FKBD, which includes the active site, showed the typical fold, which was nearly identical to the crystal structure of full-length Mip. It consisted of six β-strands, which formed an antiparallel sheet with the topology β₁-β₂-β₅-β₆-β₃-β₄. A short helix α₄ was located across this sheet. From N-terminus to C-terminus the secondary structure of Mip^77–213 included helix _α3 (N-78 to N-95), strands β₁ (V-102 to V-103), β₂ (Q-109 to N-114), β₃ (T-126 to L-135), β₄ (separated into two segments comprised of V-140 to S-143 and of A-151 to Q-154 by a bulge of seven residues), helix α₄ (P-160 to L-166) and strands β₅ (T-174 to Y-178) and β₆ (L-200 to V-209). The strands were all connected by short loops except strands β₅ and β₆ which were connected by a long hairpin loop (V-179 to T-199). Similar to human FKBP12, a hydrophobic cavity was formed in the presumed center of PPIase activity. It was located between the α₄-helix and the interior side of the β-sheet and mostly composed of hydrophobic residues. The side chains of W-162 and F-202 formed the bottom of this pocket and were surrounded by Y-131, F-141, D-142, F-153, Q-157, V-158, I-159, P-193, and I-194. The crystal structure of the Mip homodimer is only slightly different from the solution structure presented here. The root mean square deviation (rmsd) between the coordinates of the backbone without the termini (A-81 to V-209) is 0.24 nm between the two structures.

An analysis of chemical shift perturbation data from the 2D ¹⁵N-HSQC experiments of free and complexed Mip^77–213 indicated significant changes in the chemical environment of residues Y-102, D-142, T-144, F-153 to A-161, A-165, F-185, F-202, and K-203 upon binding of rapamycin. These residues were located in the hydrophobic cavity or in its direct vicinity, clearly indicating that the cleft was involved in binding rapamycin. This had already been assumed from the analysis of the structure of the homologous human FKBP12 in complex with FK506 and rapamycin. The data suggest that the loop between strand β₄ and helix α₄, where chemical shift changes were most pronounced, plays a key role in recognition of the ligand.

The average structure of Mip^77–213 in complex with rapamycin was similar to the free protein with an rmsd between the backbone coordinates of only 0.26 nm for residues A-81 to V-209 (Figure 2). Structural statistics are listed in table 2. While the secondary structure of Mip remained nearly unchanged, the overlay of the average structures showed a structural rearrangement of the bulge interrupting strand β₄ (residues T-144 to P-150) as well as of the hairpin loop (residues V-179 to T-199). Upon binding, the former was significantly displaced, enlarging the binding pocket to accommodate the ligand. A distance of 0.60 nm was observed between the positions of G-148 C_α in the free and bound average structures. Within the hairpin loop, intrinsic changes were observed. The part of the hydrophobic cavity that was formed by P-193 and I-194 in free Mip^77–213 became occupied by Y-185, for which a strong change in chemical shift had been observed. The stretch from V-190 to P-196 was bent away from the binding pocket, which was reflected in a lower rmsd of only 0.21 nm between free and complexed protein, if these seven residues were not considered. The hairpin loop and the bulge were the two regions that contributed most to the overall rmsd. Omitting these two regions, the rmsd between free and bound Mip was 0.17 nm for the backbone and 0.15 nm for the secondary structure elements only. The overlay of an ensemble of 16 refined complex structures hinted at flexibility in these two sections. The hairpin loop appeared slightly flexible in the simulations, with a more stable N-terminal part (Figure 3).

A comparison with the crystal structure revealed that the orientation of the hairpin loop in the Mip^77–213-rapamycin complex was nearly identical to the orientation in free, full-length Mip. This similar configuration resulted in a backbone rmsd value of 0.15 nm between the two structures for residues A-81 to V-209. In the solution structure of free Mip^77–213, the orientation of the hairpin loop was different. Y-185, which formed the outer edge of the binding pocket in the crystal structure and in the complex, was displaced, and its position occupied by the residues P-193 and I-194 (Figure 4). This structural rearrangement in Mip^77–213 may be an artifact due to the lack of the dimerisation domain. In the crystal structure, the connecting α-helix was stabilized by the hairpin loop via side chain hydrogen bonds withdrawing the residues P-193 and I-194 from the hydrophobic cavity. In the mutant, high flexibility of the N-terminus may have rendered side chain interactions in this part of the helix unfavorable and caused the reorientation of the loop. However, for full-length Mip in solution high flexibility of the hairpin loop was observed by NMR relaxation measurements 7. Apart from the hairpin loop, all three structures superimposed very well. Without the loop, the rmsd values between the crystal structure and either free or bound Mip were similar (0.17 nm and 0.15 nm, respectively).

To further investigate the stabilization of the hairpin loop, heteronuclear relaxation rate constants R₁ and R₂ and Nuclear Overhauser Effects (hetNOE) were measured for rapamycin-bound Mip^77–213 and compared to those for the free protein 7 (Figure 5). As had also been observed for free Mip, the relaxation data indicated the presence of a stable secondary structure in the complex. HetNOE values < 0.65, indicating the presence of fast motion on a picosecond timescale 20, were observed for most of the bulge residues (K-146 to K-149) in the complex. This observation provided further evidence for flexibility and fast motion, in accordance with the results of the structure calculations. In free Mip^77–213, similar values were found for these residues, indicating that the local dynamics of the bulge were not restricted by the presence of rapamycin. Small differences (hetNOE values were slightly lower in the complex) may be due to the structural reorientation of the bulge. Different observations were made for the hairpin loop. For residues R-188 to G-192 of the free enzyme, hetNOEs were smaller than 0.65 and the R₁/R₂ values were elevated. Upon binding of rapamycin, NOE values were larger for these residues and R₁/R₂ values were not elevated. These differences reflect a decreased flexibility in this part of the hairpin loop in complexed Mip as compared to the free protein. The overall correlation time τ_cee was derived from the measured R₁/R₂ ratios assuming isotropic tumbling. The correlation time was 8.3 ns for free Mip and 11.6 ns for the Mip-rapamycin complex. In order to assess whether this large change is in agreement with the solution structure of the complex, the correlation time for molecular tumbling was calculated from the expected hydrodynamic radius using HYDRONMR 21. τ_c values of 9.7 ns and 10.8 ns were obtained for free Mip and the complex, respectively, confirming that molecular tumbling was considerably slowed down by the increased hydrodynamic radius of the complex. The larger increase observed experimentally was most likely due to higher rigidity of the complexed protein, which was not considered in the theoretical model.

Rapamycin was bound to Mip with the pipecolyl ring (C2–N7, see Figure 1 for nomenclature) penetrating deep into the hydrophobic cavity. The ring was surrounded by the aromatic side chains of Y-131, F-153, W-162, and F-202 as well as by residues V-158 and I-159. Intermolecular NOEs were observed for all of these residues except for F-202. The binding domain of rapamycin is comprised of the ester linkage, the pipecolyl ring, the dicarbonyl group, and the pyranosyl ring. The stretch from C14 to C24 was fully exposed, while the cyclohexyl ring was partly accessible to the solvent. Rapamycin was not as well-defined as Mip^77–213, due to the lack of intramolecular distance restraints. This fact was expressed by a higher average rmsd of the coordinates of all heavy atoms for rapamycin (0.15 ± 0.03 nm) than for the whole complex including the protein (0.081 ± 0.007 nm).

Between Y-185 OH and the rapamycin carbonyl group at C8, an intermolecular hydrogen bond was observed in all of the calculated ensemble structures. Another hydrogen bond involved Y-185 OH and N7. However, there were more possible acceptors for the hydrogen of Y-185 OH at the inner side of the macrolide ring pointing towards the protein in the ensemble. Another hydrogen bond was formed by Y-131 OH and the carbonyl group at C9 of rapamycin. Intermolecular contacts were also found for both oxygen atoms of residue D-142 and the OH-group at C10.

It has been demonstrated by Wintermeyer et al. that both D-142-L and Y-185-A mutations resulted in strongly reduced PPIase activity of the recombinant Mip proteins (5.3 and 0.6% activity compared to wild-type Mip, respectively) 22. In the complex, both amino acids were observed to be within the hydrophobic cavity and to form hydrogen bonds stabilizing the Mip-rapamycin complex. Since binding of rapamycin efficiently inhibits PPIase activity 8, the hydrophobic cavity of the protein is most likely the active site of the enzyme.

The importance of the residues involved in binding of rapamycin is confirmed by their good or strict conservation in species of different kingdoms (Figure 6). Galat has investigated FKBPs with a molecular weight of about 12 kDa and 13 kDa from diverse organisms 23. The residues corresponding to Y-131, F-141, D-142, F-153, V-158, and W-162 in Mip are well conserved in all sequences among the two groups. I-159, Y-185, and F-202 are even strictly conserved. Except for Y-185, which plays a key role in binding of rapamycin, these residues form the hydrophobic cavity of free Mip^77–213. Additional residues of Mip involved in binding without being conserved are Q-157, I-194, and especially P-193, which is rarely observed at this position in other FKBDs. The two amino acids of the flexible loop P-193 and I-194 form part of the hydrophobic cavity in free Mip^77–213, while Q-157 is located on its edge.

The homology of human FKBP12 and L. pneumophila Mip is reflected in a high degree of similarity of their hydrophobic cavities. This cavity is formed by the residues Y-26, F-36, D-37, F-46, E-54, V-55, I-56, W-59, Y-82, and F-99 in FKBP12. All residues occupy identical positions as their counterparts in Mip, where F-46 is the only exception. The functional analogue in Mip is F-153, while the corresponding sequence position is occupied by A-151. Interestingly, the sequence position corresponding to F-153 in Mip is occupied by F-48 in FKBP12, which does not directly contribute to binding in the FKBP12-drug complexes. This functional substitution forces a rotation in the side chain of F-153 by about 100° as compared to F-48 in the crystal structure of the FKBP12-rapamycin-complex (Figure 7). The orientation of F153 was experimentally well defined by a total of 73 intramolecular (non-intraresidual) NOEs and 42 intermolecular NOEs to rapamycin. Apart from FKBP12, there are other FKBDs with a triad -FXF- at this sequence position, which is either substituted by -XXF- or -FXX- among other representatives of this group of proteins (Figure 6). This might represent an example of a compensatory mutation 24 during the evolution of the FKBDs. Interestingly, the conformation of the side chain of F-48 in the FKBP12-rapamycin complex is similar to that of one of F-153 in the solution structure of free Mip^77–213, while the side chain conformation of F-153 in the Mip^77–213-rapamycin complex is similar to the crystal structure of free Mip (Figure 8). For the secondary structure of bound Mip this has the consequence that the C-terminal segment of strand β₄ is slightly steeper than in free Mip or in bound FKBP12 (Figure 8). This difference explains the observed displacement of the bulge of sheet β₄. A further structural difference between the two proteins is that Y-82 forms part of the hydrophobic cavity in both the crystal 25 and the solution structure 26 of free FKBP12. The respective counterpart in Mip is Y-185, which is part of the binding pocket only in the crystal structure, and replaced by P-193 and I-194 in the solution structure. As a consequence, structural rearrangements upon binding with rapamycin are less pronounced in FKBP12 2728 than for Mip (Table 3).

Previous NMR investigations of the FKBP12-FK506 29 complex revealed that, in contrast to the uncomplexed FKBP12, the residues Y-82 to H-87 of the hairpin loop (P-78 to A-95) were rigidly fixed. The flexibility was reduced due to stabilizing interactions by the side chains of H-87 and I-91, as well as by a hydrogen bond from Y-82 to the C8 carbonyl of FK506. The conclusion that Y-82 is a key residue in FKBP12 was further supported by its substitution with leucine 30. The results for the Mip^77–213-rapamycin complex are completely analogous to these observations. Y-185, the counterpart of Y-82 in FKBP12, plays the same role in the hairpin loop in Mip. Since this is a common scheme in both proteins, flexibility of the loop may be crucial for the recognition of the protein targets calcineurin and mTOR, respectively, and also for selectivity of the binding.

The high structural similarity between the Mip-rapamycin and FKBP12-rapamycin complexes suggests that FKBP12 ligands are suitable leads for drug design. Rapamycin itself appears as a promising starting point for two reasons. First, rapamycin is an approved and widely used drug, making undesired side effects of its derivatives less probable than for totally new agents. Second, substances based on rapamycin have the potential to be highly active against Legionnaires' disease, because unmodified rapamycin is a subnanomolar inhibitor of FKBP12 3132 (K_i = 0.2 nM) and on the other hand has been shown to inhibit penetration of a lung epithelial barrier by L. pneumophila in vitro 3. The immunosuppresive properties of novel derivatives may be avoided according to the dual domain concept, which implies separate FKBP binding and effector domains in the drug. Immune modulation is mediated by binding a target protein to the effector domain. In the Mip-rapamycin complex molecular contacts are found exclusively to the FKBP binding domain of rapamycin, suggesting that the removal of the effector domain would not influence affinity. Inhibitors composed only of the FKBP binding domain but lacking the effector domain were suggested to have no influence on the immune response. Drug molecules such as, for example, the sub-nanomolar inhibitor V-10,367 (K_i = 0.5 nM) 33, or a series of sub-micromolar inhibitors of FKBP12 34, do not affect the immune response or calcineurin activity 35, respectively. However, it has been shown that FK506 (K_i = 0.6 nM for FKBP12) 31 as well as V-10,367 foster nerve regeneration in SH-SY5Y neuroblastoma cells 36. These side effects call for further investigations and clinical trials before a novel drug may be approved.

Escherichia coli harboring a plasmid encoding the PPIase domain of Mip and part of the connecting α-helix were used to overproduce Mip^77–213 in ¹³C- and ¹⁵N-labeled medium (Martek M9). The enzyme was purified from these bacteria as described previously 2. The NMR sample contained 2.5 mM of double labeled (98% ¹³C, 98% ¹⁵N) Mip^77–213 dissolved in 20 mM potassium phosphate buffer in 90% H₂O and 10% D₂O at pH 6.5. For the experiments with the complex, unlabeled, dry rapamycin (LC Laboratories, Woburn, USA) was added to the protein solution until the non soluble drug precipitated. The solution containing a 1:1 complex of rapamycin and Mip^77–213 was then centrifuged.

The NMR experiments were performed on Bruker Avance 600, 700, and 800 MHz spectrometers at a temperature of 298 K. X-filtered spectra of the complex were acquired on a Bruker Avance 700 with CryoProbe. The data were processed using NMRPipe 37. Sequence-specific backbone and side chain resonance assignments of free Mip^77–213 were obtained as described previously 38. Assignments for the backbone resonances of rapamycin-bound Mip^77–213 were analyzed using 3D HNCO, HNCA, HNCACB, and CBCA(CO)NH spectra. HNHA, HBHA(CO)NH, C(CO)NH and HCCH-TOCSY were used for aliphatic side chain ¹H and ¹³C assignments. Assignments for the amino groups were obtained by 3D CBCA(CO)NH, 2D HSQC and 3D ¹⁵N-NOESY spectra. The aromatic ¹³C resonances were assigned from ¹⁵N-edited NOESY, ¹³C 2D HSQC and 3D NOESY centered at the aromatic frequency. For the assignments of intermolecular distance restraints ¹³C-filtered, ¹³C-edited 3D NOESY spectra were recorded separately for aliphatic and aromatic ¹³C resonances. Both peak picking and visualization of the spectra were performed using NMRView 39.

The ¹H-¹⁵N relaxation experiments were performed at 600 MHz, using pulse sequences from Dayie and Wagner 4041. The R₁ values were determined by performing 11 experiments with eight different delays [5.38 (two times), 32.20, 64.38, 128.73 (two times), 300.35, 697.21 (two times), 1297.87, and 1995.06 ms]. To determine the R₂ values, 11 experiments with five different delays [16.96 (two times, 50.84 (two times), 101.65 (three times), 152.47 (two times), and 203.28 ms (two times)] were performed. For the measurement of the heteronuclear ¹H-¹⁵N NOE, the relaxation delay was set to 4 seconds. Proton saturation was achieved by applying 600 high-power pulses with an interpulse delay of 5 ms for the final 3 s of the relaxation delay in the saturation experiment. Correlation times (τ_c) averaged over different regions of the protein were calculated using the TENSOR2 software package 4243 assuming isotropic tumbling. Only relaxation rates of residues showing a HetNOE of > 0.65 were used. HYDRONMR 21 was used to estimate the correlation times (τ_c) from the atomic coordinates files.

Structure calculations were performed on Opteron based multi-core compute servers with 16 GB RAM under Linux. The structure of free Mip^77–213 was calculated with ARIA/CNS 4445 using 1737 unambiguous and 784 ambiguous NOE distance restraints (Table 1). The backbone dihedral angle restraints for the structures of Mip^77–213 were determined with TALOS 46. Calculation of the complex structure was carried out in two steps. First, the structure of rapamycin-bound Mip was calculated with both ARIA/CNS and Xplor-NIH 2.16 4748, using 1692 unambiguous and 2509 ambiguous NOE distance restraints (Table 2), which corresponded to 6720 ambiguous atom-to-atom distances in the Xplor-NIH calculations. The backbone rmsd between the average structures obtained with ARIA/CNS and Xplor-NIH was 0.070 nm (0.055 nm for the binding pocket), showing that the different programs introduced only minor deviations compared to the structural differences originating from binding of rapamycin (Table 3).

In the second step, the final average structure of rapamycin-bound Mip was used as input for the calculation of the complex with rapamycin. This significantly decreased the required computing time compared to random structure starting coordinates. Parameters for rapamycin were created by PRODRG2.5 49 from the crystal structure of its complex with human FKBP12 27. To facilitate first assignments of intermolecular NOEs rapamycin was placed manually in the putative binding pocket of Mip. This system was solvated in a periodic box with 10774 SPC water molecules 50 and a 3 ns molecular dynamics run was executed using GROMACS 3.3 515253 as described previously 7. The resulting model was solely used in the first assignment round and not for any further structure calculations.

Complex structures were calculated with Xplor-NIH 2.16 using intra- and intermolecular distance restraints starting with molecules separated by more than 7 nm. The resulting complex structures were compared to the NOEs in the spectra. Reassignment and new calculations were performed in an iterative fashion. In each step, new intermolecular distances were taken into account and the distances violated the most were reassigned or removed. After 14 iterations, 179 intermolecular NOEs were assigned. Refinement of the energetically lowest complex structure after the final run was performed using the modified example script for protein G of Xplor-NIH. Out of the 80 calculated structures, the 16 lowest energy structures were selected for analysis with PyMOL 54 and PROCHECK 55. All graphical representations of structures were generated using PyMOL.

A distance of less than 0.36 nm between donor and acceptor was assumed to be sufficient for an intermolecular hydrogen bond in the Mip^77–213-rapamycin structures. Thermal noise in the ensemble and the flexibility of residues T-144 to P-150 and A-179 to E-199 caused slight differences in the 16 refined structures. Hydrogen bonds were therefore only accepted if they occurred in at least half of the individual structures.

The sequences were aligned with the ClustalW 56 program and arranged with ClustalX 57.

The coordinates of the structures of free Mip^77–213 and the Mip^77–213-rapamycin complex have been deposited at the protein data bank (accession codes 2uz5 and 2vcd, respectively) 58. Chemical shifts are available at BMRB database (accession code 6334 and 15507, respectively).