Structural basis of conformational transitions in the active site and 80′s loop in the FK506-binding protein FKBP12

The extensive set of NMR doublings exhibited by the immunophilin FKBP12 (FK506-binding protein 12) arose from a slow transition to the cis-peptide configuration at Gly89 near the tip of the 80′s loop, the site for numerous protein-recognition interactions for both FKBP12 and other FKBP domain proteins. The 80′s loop also exhibited linebroadening, indicative of microsecond to millisecond conformational dynamics, but only in the trans-peptide state. The G89A variant shifted the trans–cis peptide equilibrium from 88:12 to 33:67, whereas a proline residue substitution induced fully the cis-peptide configuration. The 80′s loop conformation in the G89P crystal structure at 1.50 Å resolution differed from wild-type FKBP12 primarily at residues 88, 89 and 90, and it closely resembled that reported for FKBP52. Structure-based chemical-shift predictions indicated that the microsecond to millisecond dynamics in the 80′s loop probably arose from a concerted main chain (ψ88 and ϕ89) torsion angle transition. The indole side chain of Trp59 at the base of the active-site cleft was reoriented ~90o and the adjacent backbone was shifted in the G89P crystal structure. NOE analysis of wild-type FKBP12 demonstrated that this indole populates the perpendicular orientation at 20%. The 15N relaxation analysis was consistent with the indole reorientation occurring in the nanosecond timeframe. Recollection of the G89P crystal data at 1.20 Å resolution revealed a weaker wild-type-like orientation for the indole ring. Differences in the residues that underlie the Trp59 indole ring and altered interactions linking the 50′s loop to the active site suggested that reorientation of this ring may be disfavoured in the other six members of the FKBP domain family that bear this active-site tryptophan residue.


INTRODUCTION
The peptide prolyl isomerase FKBP12 (FK506-binding protein 12) was initially characterized for its role in binding the immunosuppressants FK506 and rapamycin. The ternary complex of FKBP12 and FK506 with calcineurin inhibits the enzymatic activity of this protein phosphatase, blocking a key T-cellactivation pathway involved in tissue transplant rejection [1]. The binding of FKBP12 and rapamycin to mTOR (mammalian target of rapamycin) inhibits its role in regulating cell growth and cancer progression [2]. Despite considerable effort directed towards developing active-site inhibitors of mTOR, the only two clinically approved therapeutics for this protein kinase are allosteric inhibitors derived from rapamycin [3].
The challenge of FKBP12 pharmacology is increased markedly by the presence of 22 homologous FKBP domains within the human genome [4]. In addition to the closely homologous FKBP12.6 and the more evolutionarily divergent FKBP13, there are 19 different FKBP domains that are modules within larger proteins. In addition to structural similarities, there is also some degree of functional overlap among these FKBP domain proteins. The mTOR interactions of FKBP12 can be functionally replaced in cellular model systems by the larger FKBP domain proteins FKBP51 and FKBP52 [5]. In a similar fashion, siRNA studies indicate that FKBP12 contributes to ∼ 60 % of the FK506-mediated inhibition of calcineurin, whereas the rest of the inhibitory effect is contributed nearly equally by FKBP12.6 and FKBP51 [6].
Although the pharmacological utility of the bacterial macrolides FK506 and rapamycin is well established, the physiological relevance of their interactions is less clear. Despite more than two decades of research, no endogenous smallmolecule ligand has been shown to mediate protein-protein interactions for the FKBP domains [7]. Both biochemical [8,9] and genetic [10,11] evidence support the importance of the binding of FKBP12 and FKBP12.6 to the RyR (ryanodine receptor) Ca 2 + channels in cardiac and skeletal muscle in the regulation of contraction, as well as binding to the RyR channels of pancreatic islet cells for the regulation of insulin secretion [12] and to RyR channels in the central nervous system that are involved in memory processing [13] and in stress-induced cognitive dysfunctions [14]. FKBP12 has also been implicated in the regulation of a number of membrane-bound hormone receptors [15].
The prolyl isomerization activity of FKBP12 has not been shown to participate in any of its protein signalling interactions, and a similar lack of dependence on catalytic activity has been observed for the well-studied glucocorticoid receptor interactions of the closely homologous FKBP domains of FKBP51 and FKBP52 [16]. On the other hand, prolyl isomerization activity does appear to play a role in the association of FKBP12 with aggregated α-synuclein in the Lewy body deposits of patients with Parkinson's disease [17], with the conformationally disordered tau protein in the neurofibrillary tangles of Alzheimer's disease [18] and in the binding to the amyloid precursor protein [19]. FKBP12 has been shown to accelerate the aggregation of α-synuclein in vitro [20] and in vivo [21].
Recently, we characterized a previously unreported slow conformational transition for FKBP12 in which a minor state population of 12 % interchanges with the major conformational state at a rate of ∼ 0.05 s − 1 at 25 • C [22]. Although the primary site of the conformational transition appears to lie at the tip of the 80 s loop, which spans between the last two strands of the central βsheet, the doubling of the backbone amide resonances that arises from the two slowly interchanging conformations extends not only to residues lining the active-site cleft, but beyond to a number of residues in and surrounding the 50 s loop on the opposite side of the protein. Interestingly, the major state, but not the minor state, of this slow transition also exhibits linebroadening of the amide resonances in the 80 s loop, indicative of conformational exchange in the microsecond to millisecond timeframe. Evidence for conformational flexibility within the 80 s loop gains particular relevance due to the fact that this loop provides a major proportion of the interprotein interactions for each of the four distinct proteinprotein complexes that have been structurally determined for FKBP12 [23][24][25][26]. The homologous loop has also been shown to provide critical protein-recognition interactions in other FKBP domain proteins [16].
In the present study we have analysed the structural basis of the slow resonance doubling transition of FKBP12 and the more rapid conformational linebroadening transition in the 80 s loop to gain insight into how these effects are propagated through the protein structure. In turn, these studies have led to the characterization of a well-populated conformational transition within the active-site cleft.

Protein preparation
Genes for the wild-type and G89A and G89P variants of the human FK506-binding protein FKBP12 were synthesized chemically (Genscript) from the wild-type gene sequence, with codon optimization for expression in Escherichia coli cells. The genes were cloned into the expression vector pET11a and then transformed into the BL21(DE3) strain (Novagen) for expression. The protein expression and purification procedure for the FKBP12 proteins were carried out as described previously [22,27].
All isotopically labelled samples were prepared via protein expression in minimal medium containing 0.1 % 15 NH 4 Cl as a nitrogen source [28]. For U-13 C, 15 [29] were supplemented into a medium for U-2 H, 15 N-enriched sample growth as described previously [28].
For the protein samples expressed in 2 H 2 O medium, 1 mM tris(2-carboxyethyl)phosphine and solid Tris base was added to a solution of the purified protein to obtain a pH value above 9, and the samples were incubated at 25 • C for 3 h and then neutralized with solid monobasic sodium phosphate. All protein samples were concentrated via centrifugal ultrafiltration (1000 g for 30 min at 4 • C) and then equilibrated into a pH 6.50 buffer containing 25 mM sodium phosphate, 2 mM DTT and 2 mM tris(2-carboxyethyl)phosphine by a series of centrifugal concentration steps (1000 g for 30 min at 4 • C). For the crystallization trials, the protein sample was neutralized and then equilibrated into 5 mM sodium chloride and concentrated by centrifugal ultrafiltration (1000 g for 30 min at 4 • C).

X-ray crystallography
Crystals of the C22V,G89P variant of FKBP12 were grown at room temperature (22 • C) in hanging drops by microseeding from a crushed crystal of the FKBP C22V,H87V variant [22]. Before microseeding, 2 μl of protein solution at 22 mg/ml concentration was mixed with an equal volume of reservoir solution containing 64 % saturated ammonium sulfate, 0.1 M Hepes (pH 7.5) and 4 % methyl-2,4-pentanediol, and equilibrated overnight. The crystals belonged to space group C2 and had the cell parameters a = 71.37 Å, b = 35.86 Å, c = 41.24 Å and β = 96.65 • . There was one molecule per asymmetric unit with a crystal solvent content of 48 %. Before data collection, crystals were gradually transferred into a reservoir solution containing a higher concentration of ammonium sulfate (up to 80 %) at 5 % per step, and then flash-cooled under a nitrogen stream at 100 K and stored in liquid nitrogen. Diffraction data were collected at 100 K using an RAxisIV + + detector and an inhouse Rigaku microfocus Micromax-007 X-ray generator. These diffraction data were processed and scaled using CrystalClear 1.3.6 (Rigaku). Subsequently, higher-resolution diffraction data were collected at 100 K using beamline X25 of the National Synchrotron Light Source (Brookhaven National Laboratory). These data were processed and scaled using HKL2000 [30]. With the high-resolution structure of FKBP12 [31] (PDB code 2PPN) used as a search model, clear solutions were found with the PHASER molecular replacement program within the PHENIX suite [32]. Structural refinement was carried out using PHENIX and SHELX-97 [33]. Model rebuilding was carried out using Coot [34]. Figures of crystallographic structures were generated using the Chimera software [35].

NMR spectroscopy
NMR data for the G89A and G89P variants of FKBP12 were collected on a Bruker Avance III 600 MHz and 900 MHz spectrometers at 25 • C. Backbone resonance assignments were carried out using standard HNCO [36], HN(CA)CO [36], HNCACB [37] and HN(CO)CACB [38] experiments. The resonance assignments for the wild-type protein have been reported previously [22] (Biological Magnetic Resonance Data Bank entries 19240 and 19241). The cis-glycine linkage analysis and the indole ring reorientation analysis were carried out using 3D 13 C-13 C-1 H NOESY and 13 C-15 N-1 H NOESY [39] experiments respectively. FELIX software (Felix NMR) was used for the NMR data processing. Relaxation analysis of the 15 N indole resonance of Trp 59 utilized a CSA (chemical shift anisotropy) value of − 126 p.p.m. [40].
The initial rate of NOE build-up between an isolated proton pair is proportional to: where ω is the 1 H Larmor frequency and J ij (X) are the components of the spectral density function. If the correlated internal motion of the two nuclei dissipates rapidly as compared with the rate of global molecular tumbling, the internal motion correlation function can be factored out as a generalized order parameter (S) [41] which for a three state methyl jump model can be represented as: where r j is the distance between the methyl proton j and a distal proton and θ ij is the angle between the vectors formed between a distal proton and the two methyl protons i and j. Cross-peaks from the G89P variant are indicated in red. Resonances that are doubled in the wild-type spectrum are identified. The Ala 84 resonances were not observed in this spectrum due to severe linebroadening presumably because of the rapid amide hydrogen exchange as observed in FKBP12 [27]. f , folded side chain resonances.

Mutational variation of the slow exchange conformational equilibrium in FKBP12
The largest chemical-shift differences for the doubled amide resonances of FKBP12 are seen for residues in the 80 s loop [22]. The resonance doubling extends into the first two residues of the β 5 -strand and the structurally contiguous residues of the adjacent β 2 -and β 3α -strands ( Figure 1). The doubling also extends backwards along the chain into the 3 10 turn and into the nearby α-helix which bears the Trp 59 indole side chain that forms the base of the active-site cleft. The residues exhibiting resonance doubling extend beyond the active site to the beginning of the 50 s loop that abuts the C-terminal end of the α-helix. We reported previously the crystal structure and 15 N relaxation analysis of an H87V variant of FKBP12. Despite the close similarity of the H87V structure [31] (0.25 Å C α RMSD with respect to the 0.92 Å wild-type structure), the slow resonance doubling transition is completely quenched throughout the protein [22]. Chemical shift-based prediction of the backbone torsion angles for the minor slow exchange state yielded a negative φ torsion angle for Gly 89 as compared with a value of + 103 • reported in the 0.92 Å resolution structure of FKBP12 [31] (PDB code 2PPN).
In the present study, the G89A mutation was introduced so as to shift the conformational equilibrium towards a negative φ value for this residue. The 2D 1 H, 15 N HSQC spectrum of the G89A variant yielded a pattern of amide resonance doublings with chemical-shift values quite similar to that observed for the wildtype protein [22], although the relative intensity of cross-peaks from the two slow exchange conformational states is markedly different (Supplementary Figure S1 at http://www.biochemj.org/ bj/458/bj4580525add.htm). In the wild-type spectrum the minor slow exchange state represented only 12 % of the total population, whereas in the G89A variant the analogous set of cross-peaks yielded 67 % of the protein signal. This change in population corresponded to a 6.7 kJ/mol difference in the relative stability of the two conformations that was induced by the alanine residue substitution.
To examine further how enforcing a negative φ torsion angle at residue 89 affects the structure and dynamics of FKBP12, the G89P variant was analysed by NMR. The proline residue substitution eliminates peak doubling for all of the amide resonances ( Figure 1). The similarity in chemical-shift behaviour for the G89P variant and the minor slow exchange conformation of the wild-type protein indicated strongly a corresponding similarity in structure. The only marked differences in the superimposition of these two sets of resonances occurred at residue 89, for which the G89P variant lacked an amide resonance, and Ile 90 for which the 15 N of the G89P was shifted downfield as anticipated from the inductive effects resulting from the side chain substitution for the preceding residue [42,43].

Crystal structure for a G89P variant of FKBP12 at 1.50 Å resolution
Taking advantage of the close structural similarity between the wild-type and the evolutionarily conservative C22V variant of FKBP12 and the increased stability of the cysteine residuefree variant [22], the G89P mutation was introduced into the C22V background for crystallization trials. Crystals were grown in the C2 space group using microseeding from crystals of the C22V,H87V variant [22]. Diffraction data were collected to 1.50 Å resolution using an in-house diffractometer. Following refinement, the backbone φ and ψ torsion angles for Pro 89 were found to be ( − 92 and − 12) with the φ value being nearly 180 • from that of Gly 89 in the wild-type structure. Furthermore, this crystal structure adopted a cis-peptide conformation for the linkage between Pro 88 and Pro 89 . Superimposition with the high-resolution wild-type structure indicated that, although the conformation of residues 89 and 90 differed markedly, the residues preceding Pro 88 and those following Ile 90 aligned closely ( Figure 2A).
The sequence at the tip of the 80 s loop differed by only one residue between the first FKBP domain of FKBP52 (PPKIPP) and the G89P variant of FKBP12 (PPIIPP). A cis-peptide linkage at the position homologous with Pro 89 has been reported in crystal structures of this FKBP52 domain (e.g. PDB codes 1N1A [44] and 4LAV [45]). This region of the 80 s loop in the G89P structure superimposed closely with that of the PDB code 1N1A structure for FKBP52 ( Figure 2B).
If the minor slow exchange state of FKBP12 corresponds to the transition of the Gly 89 peptide linkage to a cis configuration, the crystal structure of the G89P variant provides a ready explanation for how the H87V substitution for FKBP12 might suppress this transition. In the crystal structure of the H87V variant, the C γ 2 atom of that valine residue is 3.7 Å from the C δ1 atom of the evolutionarily invariant Tyr 82 side chain [22]. When a valine residue side chain was modelled on to His 87 of the G89P crystal structure, the separation between the valine C γ 2 atom and the C δ1 atom of Tyr 82 was reduced to 2.9 Å, suggestive of a substantial steric hindrance to such a conformational transition.

Glycine cis-peptide configuration at residue 89 in wild-type FKBP12
If the peptide linkage of Gly 89 in wild-type FKBP12 assumes a cis-peptide linkage in the minor state of the slow exchange transition, this implies that this critical protein-recognition loop of FKBP12 adopts a FKBP52-like conformation in a substantial fraction of the conformational distribution. One indirect line of support for this possibility is the 70 kJ/mol activation energy that was determined for the slow exchange process in FKBP12 [22], which is typical for cis-trans isomerization of peptides. On the other hand, non-proline residue cis-peptide linkages are quite rare in protein crystal structures, occurring at a frequency of only 0.03 % [46]. To demonstrate directly whether the minor slow exchange state of FKBP12 adopts a cis-peptide linkage at this residue, 3D 13 C-13 C-1 H NOESY measurements were carried out to determine the presence of a short range distance between the H α atoms of Pro 88 and Gly 89 in the minor conformation. For the more common trans-peptide linkage, the distance between sequential H α atoms is ∼ 4.5 Å, nearly independent of the intervening ψ i and φ i + 1 torsion angles [47]. On the other hand, assuming that the minor slow exchange form of the wild-type protein adopts the geometry of this linkage, as found in the G89P crystal structure, the distances from the Pro 88 H α to the two Gly 89 H α atoms were 2.0 and 3.4 Å.
For the minor state resonances of wild-type FKBP12, the intensity of the sequential NOE transfer from the 1 H α resonances of Gly 89 to the 1 H α of Pro 88 was more intense than the intraresidue NOE transfer to Pro 88 1 H α from its own 1 H β

Figure 3 1D slices from the 3D 13 C-13 C-1 H NOESY spectrum of wild-type FKBP12
(A) At the 1 H and 13 C frequencies for the C α of Pro 88 in the minor slow exchange state the NOE cross-peak correlating the sequential C α of Gly 89 is more intense than the corresponding NOE cross-peak correlating the intraresidue Pro 88 C β interaction. (B) In contrast, the corresponding sequential NOE connectivity correlating the C α nuclei of Pro 88 and Gly 89 in the major slow exchange state is comparatively far less intense than the intraresidue NOE to the Pro 88 C β . resonances ( Figure 3A). In contrast, for the major slow exchange state the sequential NOE transfer from the 1 H α resonances of Gly 89 to the 1 H α of Pro 88 was much weaker than the intraresidue transfer from the 1 H β resonances ( Figure 3B). A similar pattern of NOE cross-peak intensities were observed for transfer in the reverse direction from the 1 H α of Pro 88 to the 1 H α resonances of Gly 89 for both the major and minor slow exchange states.

NMR chemical-shift analysis of the backbone conformations for the 80 s loop
Having established that the extensive resonance doubling of FKBP12 arises from the slow cis-trans isomerization of the peptide linkage between Pro 88 and Gly 89 , we then examined the structural basis for the much more rapid linebroadening conformational dynamics of the 80 s loop. The transition from the wild-type FKBP12 crystal structure to a conformation resembling the G89P crystal involved not only the change to the Gly 89 cispeptide configuration. The φ torsion angle of Gly 89 is + 103 • in the high-resolution crystal structure of wild-type FKBP12 [31], whereas the five-membered ring of the homologous Pro 120 in the FKBP52 structures forces the φ angle to be negative. To examine the plausibility that Gly 89 of FKBP12 might undergo a relatively rapid transition between positive and negative φ angle values while remaining in the predominant trans-peptide configuration, the other crystal structures of FKBP52 were considered. As noted above, two crystal structures of FKBP52 (PDB codes 1N1A [44] and 4LAV [45]) exhibit a cis-peptide linkage between Pro 119 and Pro 120 . However, there are two other crystal structures of FKBP52 (PDB codes 1Q1C [48] and 4LAW [45]) for which this peptide linkage is in a trans configuration. These latter two crystal structures of FKBP52 can provide models for the conformation of the 80 s loop of FKBP12 in which Gly 89 switches to an φ angle<0, whereas its peptide linkage remains in a trans configuration.
T 2 transverse relaxation experiments serve to measure the 15 N linebroadening effects which arise from the difference in chemical shift for a given nucleus that interchanges between conformational states in the microsecond to millisecond timeframe. A number of empirical algorithms have been developed for predicting NMR chemical shifts from protein structures [49][50][51][52]. One widely used algorithm is the SPARTA + program of Shen and Bax [49] which has applied an artificial neural network analysis against a database of 580 proteins to derive its parameterization. Consistent with the results from other such chemical-shift prediction algorithms, SPARTA + yielded an S.D. of 2.45 p.p.m. in its predictions of backbone 15 N chemical shifts for a set of 11 validation proteins, an appreciably lower precision than was obtained for the 13 C , 13 C α , 13 C β , 1 H α and 1 H N chemical shifts. In modelling the linebroadening for the 80 s loop resonances of FKBP12, the 15 N chemical shift predictions were only applied in a differential mode in anticipation of an increased precision in the resultant analysis.
Exploiting the fact that these chemical shift predictions are dominated generally by the local backbone geometry, the crystal structures for the 80 s loop in FKBP12 [31] (PDB code 2PPN) and the first FKBP domains of FKBP52 (PDB codes 1Q1C [48] and 4LAW molecules A and B [45]) were transformed to a common sequence by converting the disparate residues into alanine, except for the site of the cis-peptide transition for which the FKBP52 structures were converted into Gly 120 . The predicted 15 N chemicalshift values from the FKBP12-derived structure were compared against those from the three FKBP52-derived structures, both individually and as a simple average over those three structures. In comparing these predicted differences in 15 N chemical shift with the experimentally observed 15 N linebroadening effects [22], averaging the predictions over the three FKBP52-derived structures provided the strongest correlation ( Figure 4).
Given the substantial challenges for quantitative structurebased predictions of conformational exchange linebroadening effects, the degree of correlation between the predicted and observed linebroadening in the 80 s loop of FKBP12 suggests strongly that Gly 89 transiently adopts a negative φ torsion angle when that residue is in a trans-peptide configuration. On the other hand, the absence of 15 N conformational exchange linebroadening for the 80 s loop when Gly 89 is in the cis-peptide configuration of the minor slow exchange state [22] suggests that positive φ torsion angles for Gly 89 are not energetically accessible from that state in a comparable population or timeframe to that observed for the transpeptide configuration. The simplest mechanistic interpretation consistent with these data is that the two slowly interchanging Gly 89 peptide configuration states, monitored by the extensive Model-free [41] analysis of the R 1 , R 2 and NOE 15 N relaxation measurements was used to estimate conformational exchange linebroadening [22]. The differences in 15 N chemical shift were predicted with SPARTA + [49] applied to FKBP12 (PDB code 2PPN [31]) and the first FKBP domain of FKBP52 (PDB code 1Q1C [48] and non-equivalent monomers of PDB code 4LAW [45]). An asterisk indicates not statistical significant conformational exchange linebroadening (<∼ 0.4 Hz). The amide resonances for both Ala 84 and Gly 89 are severely broadened due to rapid hydrogen exchange [22], precluding a reliable determination of conformational exchange linebroadening, and the predicted chemical shift differences for these two residues are indicated as open bars.
resonance doubling, interchange with each other only via a more weakly populated transient state in which the φ torsion angle of Gly 89 is negative, whereas the peptide configuration remains trans.
As noted above, the H87V substitution suppresses all of the amide resonance doubling, presumably by its steric collision with the Tyr 82 ring in the Gly 89 cis-peptide configuration. This substitution also suppresses the microsecond to millisecond linebroadening transition for the resonances in the 80 s loop [22]. We propose that this linebroadening arises from a ∼ 180 • shift in the φ torsion angle of Gly 89 . The ψ angle of Pro 88/119 also shifted by an average of 130 • between the wild-type FKBP12 and three FKBP52 crystal structures in the trans-peptide configuration. These three crystal structures of FKBP52 can be used to examine this model for the quenching of linebroadening in the 80 s loop. Residue 118 (homologous with His 87 of FKBP12) was computationally transformed into a valine for each of these three FKBP52 structures. For PDB code 1Q1C and monomer A of PDB code 4LAW, this valine residue side chain must strongly overlap with either the aromatic ring of Tyr 113 (homologous with the evolutionarily invariant Tyr 82 ) or with the backbone of Ile 122 . Such a steric conflict could be expected to preclude the transition of Gly 89 to a negative φ torsion angle in the H87V variant, thus eliminating the linebroadening effect for the 80 s loop. A caveat to this detailed structural interpretation is that in the significantly differing local geometry of monomer B in the PDB code 4LAW structure of FKBP52 the introduction of the valine residue side chain at residue 118 does not appear to yield a severe steric conflict.
Both the H87V [53] and G89P [54] variants have been shown to have an equivalent peptide isomerase activity with that of wild-type FKBP12 in the standard assay using blocked tetrapeptides. The H87V variant appeared to block the resonance doubling and the conformational linebroadening of the 80 s loop by constraining Gly 89 to remain in the trans-peptide configuration with a positive φ torsion angle. The G89P variant appeared to block the resonance doubling and the conformational linebroadening of the 80 s loop by constraining residue 89 to remain in the cis-peptide configuration with a negative φ torsion angle. The linebroadening transition and the much slower resonance doubling transition did not appear to participate in the peptide isomerization activity of this enzyme, at least for simple conformationally unstructured peptides. Any functional relevance of this conformational plasticity/dynamics may instead relate to a role in the protein-recognition interactions of FKBP12.

Perpendicular reorientation of the Trp 59 indole at the base of the active-site cleft in a G89P variant of FKBP12
The most unexpected aspect of the G89P crystal structure was that the indole ring of Trp 59 at the base of the active-site cleft was oriented perpendicular to the conformation observed in each of the more than 30 crystal structures of wild-type FKBP12 ( Figure 5A). The aromatic rings of Tyr 26 , Phe 46 , Phe 48 and Phe 99 and the side chains of Val 55 and Ile 56 form the walls of the active-site cleft surrounding the Trp 59 ring. The reorientation of the indole ring in the G89P crystal structure occluded much of the volume of the active-site cleft, conflicting with the binding geometry observed in the crystal structures of the FK506-and rapamycin-ligated proteins. As compared with the wild-type crystal structure, the aromatic rings of Phe 48 and Phe 99 in the G89P structure appeared to move slightly inward towards the reoriented indole ring. The conformation of the residues that lie behind the Trp 59 indole ring was essentially identical for the wild-type and the G89P structures. A small cavity lies beneath the indole ring in the wildtype structure, and the nearby side chains of Val 24 , Val 63 , Leu 74 and Val 101 were essentially unperturbed by the rotation of the indole ring ( Figure 5B).
This perpendicular reorientation of the Trp 59 aromatic ring in the G89P crystal structure was strikingly similar to that reported for the E60Q (PDB code 2PPP) and E60A (PDB code 2PPO) variants of FKBP12 [31]. In addition, the backbone of the helical residues surrounding Trp 59 in the G89P crystal structure was closely similar to that observed in the E60Q (and E60A) structure ( Figure 5D). In contrast, the hydrogen-bonding interactions for Trp 59 O to Val 63 H N and Glu 60 O to Ala 64 H N were significantly irregular in the crystal structure of wild-type FKBP12 with the carbonyl groups being shifted laterally by over 1 Å with respect to the amide bond vectors as compared with the canonical hydrogen-bonding geometry seen in the G89P structure ( Figure 5C). Regarding the role of crystal packing interactions in the present study, it may be noted that the P2 1 crystal forms for the 0.94 Å resolution structure of the E60Q variant and the 1.29 Å resolution structure of the E60A variant were isomorphous with that used in the 0.92 Å resolution structure of wild-type FKBP12 [31]. The G89P variant crystallized in the C2 space group.
Saven and colleagues noted that the Glu 60 side chain in their high-resolution structure of wild-type FKBP12 extends towards the backbone of the 50 s loop to co-ordinate the amide hydrogens of Gly 51 and Gln 53 , as well as to a highly conserved solventinaccessible water molecule [31]. Introduction of glutamine at position 60 of FKBP12 results in the flipping of the peptide unit that links Lys 52 and Gln 53 and a shift in co-ordination for the buried water molecule. Although the backbone of the 50 s loop in the E60A variant remains similar to that of the wild-type protein, the side chain interaction of residue 60 with the buried water molecule is obviously disrupted. The authors [31] argued that these altered interactions of residue 60 with the buried water molecule results in a shift in the α-helix, which, in turn, presses the indole ring more tightly against the ring of Phe 99 thus driving the reorientation of the Trp 59 side chain.
The backbone of the 50 s loop and the position of the buried water molecule in the crystal structure of the G89P variant closely superimposed with that of wild-type FKBP12 ( Figure 6A). The carboxy O ε1 atom of Glu 60 preserved its hydrogen-bonding interactions with the amide hydrogens of Gly 51 and Gln 53 and with the buried water molecule. The similarity in the interactions of the Glu 60 side chain with the backbone of the 50 s loop and the buried water molecule for the wild-type and G89P structures suggested that disruption of these interactions were sufficient, but not necessary, to shift the backbone of Trp 59 and Glu 60 into a canonical α-helical hydrogen-bonding geometry and enable the reorientation of the indole ring.
The most notable difference for Glu 60 between the crystal structures of wild-type FKBP12 and the G89P variant is the transition of the side chain from a gauche − to a trans χ 1 torsion angle, which allows the backbone atoms of Trp 59 and Glu 60 to shift towards the active site indole ring without disrupting the hydrogen-bonding interactions described above. Such a shift from a gauche − to a trans χ 1 torsion angle for the Glu 60 side chain was reported previously in the crystal structure of the W59F variant of FKBP12 [55]. In this case, the backbone atoms of Phe 59 and Glu 60 underwent a similar shift to that observed in the structure of the G89P variant (Figure 6B). On the other hand, the smaller phenyl side chain at residue 59 does not become tightly pressed against the aromatic ring of Phe 99 when it maintains an orientation along the bottom of the active-site cleft similar to that of the tryptophan ring in the wildtype FKBP12 structure. In comparing the crystal structures of the W59F and G89P variants, it appeared that disruption of the steric interaction between the indole ring of Trp 59 and the phenyl ring of Phe 99 was sufficient to enable the backbone of Trp 59 and Glu 60 to shift into a canonical α-helical hydrogen-bonding geometry.
Crystal structures have been reported for four of the other six human FKBP domains which contain a tryptophan ring at the base of the active-site cleft [44,[56][57][58]. In each of these four structures, Glu 60 of FKBP12 is replaced by the shorter aspartate side chain which forms similar interactions with the backbone of the 50 s loop. It is not self-evident how these interactions could be preserved while undergoing the shift in the α-helical backbone to allow for an analogous reorientation of the tryptophan ring. A further indication that the reorientation of the active site indole side chain may be energetically disfavoured in these other FKBP domains is that the unoccupied volume beneath the plane of the Trp 59 ring in FKBP12 appears to be occluded in these other crystal structures. In the example of FKBP51 [56], the aliphatic side chains underlying the indole ring closely superimpose on those of FKBP12 illustrated in Figure 5(B), except Ile 132 which was substituted for Val 101 . This substitution resulted in the isoleucine residue C δ atom occupying the position of the void space in the FKBP12 crystal structure. Similarly, Ile 94 and Ile 105 of FKBP52 [45] replace the less bulky pair of Val 63 and Leu 74 in FKBP12, resulting in a reduced void volume beneath the tryptophan ring. If the transition of the indole ring to the perpendicular orientation is substantially more populated in FKBP12, as compared with these other FKBP domains, such a conformation might prove amenable to selective drug inhibition.

Reorientation equilibria and dynamics for the active site indole ring in wild-type FKBP12
The structural analysis of these FKBP12 variants support the possibility that there may be only a modest energy difference between the canonical and the perpendicular orientations for the indole ring of Trp 59 in the wild-type protein. Potential evidence for internal mobility of the single indole ring in FKBP12 comes from the time-resolved fluorescence anisotropy measurements of Silva and Prendergast [59] in which they derived an S 2 order parameter of 0.75 for the uncomplexed protein and an S 2 value of ∼ 1 for the FK506-bound complex. In contrast with the three fastest relaxation components for which the amplitudes and time constants were similar for the free and ligated forms of the protein, the time constant of 5.43 ns for the slowest component of the fluorescence anisotropy decay in the FK506-bound protein was decreased to 4.75 ns for uncomplexed FKBP12. The authors analysed these data assuming that this shift in the time constant arises from unresolved contributions from a local conformational transition of the indole ring and the global molecular tumbling [59], leading to a predicted time constant of 3 ns for the internal motion. In their molecular dynamics analysis on the role of the buried water molecule in modulating the conformational dynamics of FKBP12, Park and Saven [60] reported a simulation of the wild-type protein in which the Trp 59 indole ring transitioned to a perpendicular orientation after 8 ns in a 12 ns simulation. Although no reverse transition was observed, their simulation suggested the energetic accessibility of such a conformation.
In the 3D 13 C-15 N-1 H NOESY spectrum of U-13 C, 15 N-labelled FKBP12 we observed substantial cross-peaks from the indole H Nε1 of Trp 59 to both methyl groups for Val 24 and Val 63 as well as to the C γ 1 methyl of Val 101 , despite the fact that these methyls lie ∼ 7 Å from the indole H Nε1 in the crystal structure of the wild-type protein. To examine this issue in more detail, we prepared a sample of FKBP12 with 13 C enrichment for the methyl groups of valine, leucine and isoleucine residues with uniform 15 N and perdeuteration at all other carbon-bound sites as described previously by Kay and colleagues [29]. For most of the moderately intense cross-peaks of the 13 C-15 N-1 H NOESY spectra for valine, leucine and isoleucine methyl-labelled FKBP12, the NOE buildup curves were approximately linear up to a 200 ms mixing time.
A 1D 13 C slice at the 1 H and 15 N frequencies of the Trp 59 indole N-H in the major slow exchange conformational state indicates 11 peaks, most of which correspond to methyl groups that are too distant from the indole H Nε1 in the high-resolution structure of wild-type FKBP12 to give rise to the observed intensities ( Figure 7B). The analogous 1D slice at the 1 H and 15 N frequencies of the Trp 59 indole N-H in the minor slow exchange conformational state yielded a qualitatively similar pattern ( Figure 7A), although the lower sensitivity from this minor state precluded a more detailed analysis.
The methyl-indole H Nε1 cross-peak volumes were analysed to test their consistency with FKBP12 adopting a mixture of activesite conformations corresponding to the PDB code 2PPN wildtype and G89P variant crystal structures. The familiar 1/r 6 distance dependence for the NOE cross-relaxation rate typically invokes the assumption of an isolated pair of nuclear spins in the absence of internal motion.
High levels of deuteration suppress strongly spin diffusion effects thus providing a useful approximation of the isolated spin pair assumption. Furthermore, if the rapid internal motion for both of the interacting 1 H nuclei is isotropic and uncorrelated (similar to the standard assumption of crystallographic B-factor analysis for all but the highest-resolution protein structures), the resultant NOE cross-relaxation rate equals that predicted for the two nuclei being fixed at their mean relative positions [61]. Although this exact cancellation of the radial and angular dispersion effects provides a useful approximation for many types of proton pairs, the 3-fold rotational jumps of methyl protons deviates strongly from spherical isotropy. As a result, NOE calculations based on a simple 1/r 6 summation of distances from the methyl protons to a distal proton site give rise to severely  foreshortened apparent distances in protein NOESY analyses. In the present study, methyl-indole H Nε1 NOESY cross-peaks were analysed under the assumption of separability of the generalized order parameter characterizing the interaction (eqn 2) and this analysis was compared with the results derived from the more commonly used approximations of measuring NOE distances from a unified methyl group centred at either the methyl carbon or the mean position of the methyl protons. Table 1 lists the normalized experimental volumes for the methyl-indole H Nε1 NOESY cross-peaks shown in Figure 7(B). In addition to normalization against the most intense NOE resonance, the volumes for these NOE cross-peaks were normalized against the peak volumes for the corresponding methyl resonances from the 2D 1 H-13 C HSQC spectrum to compensate for differences in relaxation behaviour and in the levels of 13 C enrichment due to incomplete biosynthetic incorporation from the labelled α-oxobutyrate and α-oxoisovalerate during protein expression. Also listed are the distances from each methyl carbon to the indole H Nε1 in the PDB code 2PPN wild-type and the G89P variant crystal structures. Distances beyond 7 Å were not included in the calculations and neither were the NOE cross-peaks to the Leu 74 methyls due to ambiguity in their stereochemical assignment. This residue offers minimal utility in the NOE analysis as the distance from the closest Leu 74 methyl to the Trp 59 H Nε1 is nearly the same for both the wild-type and G89P crystal structures.
The relative NOE volumes for each of these methyl-indole H Nε1 interactions were predicted from generalized order parameter calculations [41] applied to the two crystal structures. For an admixture of 80 % and 20 % for the PDB code 2PPN wildtype and the G89P conformations respectively, the observed NOE volumes were fitted to an RMSD of 0.086 with a correlation coefficient (r) of 0.976 ( Figure 8 Table  S2 at http://www.biochemj.org/bj/458/bj4580525add.htm). The markedly worse performance for the use of the mean methyl proton position reflects principally the fact that distal protons that lie along the methyl rotation axis yield elevated predictions for both the radial and angular components of the NOE interaction.
These results indicate significant conformational plasticity in the active site of FKBP12. As there is no apparent linebroadening for the resonances involved, the indole reorientation occurs presumably more rapidly than for the microsecond to millisecond conformational exchange regime. Indication of a much more rapid transition is drawn from the 15 N relaxation analysis that we reported recently for the backbone amides of FKBP12 [22]. The R 1 and R 2 relaxation rates of 1.50 s − 1 and 5.56 s − 1 and the 15 N NOE value of 0.634 at 14.1 T (600 MHz 1 H) that we observed for

Figure 8 Predicted against observed NOE volumes for the methyl crosspeaks to Trp 59 H Nε1
The experimental NOE volumes were normalized to the maximum value (Val 101 C γ 2 ) and to the volumes of the corresponding 1 H-13 C cross-peaks in the 2D HSQC spectrum to compensate for relaxation and differential enrichment effects. Generalized order parameter calculations were performed on the crystal structures of the wild-type protein and G89P variants and then weighted at 80 % and 20 % respectively.
where p a and p b are the state populations and θ is the angle between the indole N-H bond vector orientations which is 83

Å resolution
Viewed along the plane of the indole ring for the major conformer (occupancy of 0.71), electron density for the perpendicular orientation of the minor conformer is apparent readily. The carbonyl oxygen in the minor conformer of Glu 60 (occupancy of 0.34) is shifted away from the canonical α-helical hydrogen-bonding geometry seen in the major conformer and to a position close to that observed in the wild-type FKBP12 structure [31]. The contour level for the electron density grid is 0.1261 e/Å 3 = 2σ .
wild-type FKBP12 occupies in solution and the modest differences surrounding that ring in the wild-type and G89P variant crystal structures, we examined further whether the crystal structure of the G89P variant might also yield evidence for conformational heterogeneity at this site. Since the crystals for this variant diffracted well beyond the 1.50 Å resolution data that we had initially collected on our in-house diffractometer, we collected a second dataset to 1.20 Å resolution at the National Synchrotron Light Source (Table 2). During the later stages of refinement, dual conformers were allowed for both Trp 59 and Glu 60 . Minor conformers for the two residues were obtained at occupancies of 0.29 and 0.34 respectively. When viewed edge-on for the major conformer of Trp 59 , the electron density for a perpendicular orientation of the indole ring is readily apparent (Figure 9). In the minor conformer of Glu 60 , the main chain carbonyl oxygen was shifted towards the 50 s loop into the position seen in the wild-type FKBP12 crystal structure. Although the Glu 60 C γ of the minor conformer was not well fitted to the electron density, the derived side chain χ 1 torsion angle was shifted towards the orientation seen in the wild-type FKBP12 structure. The two perpendicular orientations of the Trp 59 indole ring exhibited by the 1.20 Å resolution structure of the G89P variant reinforces the observation of a small difference in free energy for those two conformations in solution. The similar populations for the minor conformers of Trp 59 and Glu 60 in this crystal structure is consistent with a concerted shift for both the indole ring orientation and the backbone hydrogen-bonding geometry for residues 59 and 60, which, in turn, appears to be at least partially coupled to a reorientation of the Glu 60 side chain.

AUTHOR CONTRIBUTION
Sourajit Mustafi was involved in the design of the study, collection and analysis of the NMR data, and writing of the paper. Jing Zhang carried out the crystallization studies. Matthew Brecher was involved in the collection and analysis of the crystallographic data. Hongmin Li supervised and participated in the crystallographic studies as well as in writing the paper. David LeMaster prepared the protein samples and participated with Griselda Hernández in the design and supervision of the study, in the collection and analysis of the NMR data, and in writing of the paper.

Figure S2 Predicted against observed NOE volumes for the methyl crosspeaks to Trp 59 H Nε1
The experimental NOE volumes were normalized to the maximum value (Val 101 C γ 2 ) and to the volumes of the corresponding 1 H-13 C cross-peaks in the 2D HSQC spectrum to compensate for relaxation and differential enrichment effects. The methyl carbon positions were used to derive NOE volume predictions from the crystal structures of the wild-type protein and G89P variants and then weighted at 82 % and 18 % respectively.

Table S1 Methyl carbon-centred NOE analysis of Trp 59 indole reorientation in FKBP12
Leu 74 was not included due to ambiguity in the stereochemical assignment of the methyl resonances. Interactions for methyl-H Nε1 distances >7 Å were not included. The ratio of volumes between the normalized NOEs for PDB code 2PPN wild-type/G89P was 0.25. Distance, distance between methyl carbon to the indole H Nε1 in the PDB code 2PPN wild-type and the G89P variant crystal structures. NOE(exp), normalized experimental volumes for the methyl-indole H Nε1 NOESY cross-peaks.

Residue
Atom

Figure S3 Predicted against observed NOE volumes for the centeroids of methyl cross-peaks to Trp 59 H Nε1
The experimental NOE volumes were normalized to the maximum value (Val 101 C γ 2 ) and to the volumes of the corresponding 1 H-13 C cross-peaks in the 2D HSQC spectrum to compensate for relaxation and differential enrichment effects. The centroids of the methyl hydrogen positions were used to derive NOE volume predictions from the crystal structures of the wild-type protein and G89P variants and then weighted at 79 % and 21 % respectively.