Crystal structure of the FK506 binding domain of human FKBP25 in complex with FK506

Abstract

Human FKBP25 (hFKBP25) is a nuclear immunophilin and interacts with several nuclear proteins, hence involving in many nuclear events. Similar to other FKBPs, FK506 binding domain (FKBD) of hFKBP25 also binds to immunosuppressive drugs such as rapamycin and FK506, albeit with a lower affinity for the latter. The molecular basis underlying this difference in affinity could not be addressed due to the lack of the crystal structure of hFKBD25 in complex with FK506. Here, we report the crystal structure of hFKBD25 in complex with FK506 determined at 1.8 Å resolution and its comparison with the hFKBD25–rapamycin complex, bringing out the microheterogeneity in the mode of interaction of these drugs, which could possibly explain the lower affinity for FK506.

Short abstract

Interactive Figure 1 | PDB Code(s): 5D75

Introduction

FK506 and rapamycin are well‐established immunosuppressive drugs that can bind to FK506‐binding proteins (FKBPs),1 a major class of the immunophilin family. The binding of these drugs with FKBPs leads to the inhibition of T‐cell proliferation and thus immune suppression.1 Human FKBP25 (hFKBP25) is the first discovered nuclear member of the human FKBP family.2 Subsequently, FKBP25 has been shown to interact with various nuclear proteins such as MDM2, HDAC1/2, YY1, Nucleolin and HMG, and these interactions play an important role in several nuclear events like histone modifications, transcription regulation, p53 signaling, and ribosome biogenesis.3, 4, 5, 6 Human FKBP25 is a 25 kDa protein which bears two domains linked by a long flexible loop.7 Its C‐terminal domain contains the well‐conserved FK506 or rapamycin binding pocket, hence termed as the FK506 binding domain (FKBD).8 The N‐terminal domain consists of a helix–loop–helix domain, which does not show significant homology to any known human protein.9 FKBPs are also known to catalyze the cis–trans isomerization of the peptidylprolyl bond, an important step in protein folding, using the same binding pocket. The binding affinity of rapamycin or FK506 to the prototypical FKBP, hFKBP12, is almost the same,10 but hFKBP25 shows a relatively higher binding affinity to rapamycin in comparison to FK506. In fact, the binding affinity of rapamycin (K _i = 0.9 nM) to hFKBP25 is comparable to other FKBPs, whereas the binding affinity of FK506 (K _i = 200 nM) is almost 200‐fold low.2 The molecular basis underlying the lower affinity binding of FK506 to hFKBP25 has remained elusive. Though the crystal structure of the FK506 binding domain of FKBP25 (hFKBD25) in complex with rapamycin has been solved long ago,8 its complex with FK506 was not available to‐date. To understand the molecular explanation for the differential binding affinity of hFKBP25 with FK506 and rapamycin, we have solved the crystal structure of hFKBD25 in complex with FK506 and compared it with its rapamycin counterpart. Our structural analysis and comparison reveal that the subtle differences of the inhibitor's interaction with the active site residues, unique to hFKBP25, could reflect in the aforementioned affinity differences.

Results and Discussions

Structure of hFKBD25–FK506 complex

The structure of hFKBD25–FK506 complex [Fig. 1(A)] has been determined at 1.8 Å resolution, enabling us to trace all FK506 atoms unequivocally in the electron density map [Fig. 1(B)]. As expected, FK506 binds to the canonical hydrophobic pocket of hFKBD25 packed by six β‐strands (β1–β6) and an α‐helix (α1), reminiscent with other FKBPs. The r.m.s. deviation of hFKBD25–FK506 complex is 0.45 Å (for 101 equivalent C^α atoms) and 0.59 Å (for 74 equivalent C^α atoms) with the hFKBD25–rapamycin (PDB ID 1PBK) and hFKBP12–FK506 (PDB ID 1FKJ) structures, respectively, indicating that the protein atoms in these complexes adopt an almost similar conformation and thus we have to look into the minor differences in the mode of interactions of their respective inhibitors to gain molecular insights toward the reason behind their affinity differences.

Figure 1.

Crystal structure of hFKBD25–FK506 complex and its comparison with the rapamycin counterpart. (A) Cartoon representation of hFKBD25–FK506 revealing the secondary structural elements and the FK506, in stick mode, occupying the active site. An interactive view is available in the electronic version of the article. (B) The 2Fo‐Fc electron density map, contoured at 1σ cut‐off, for the FK506 molecule (shown in stick mode). (C) The interactions made by FK506, shown in light green colored stick mode, with the active site residues, shown as sticks. The residues making hydrogen bonded (along with their distances) and nonbonded interactions are shown in grey white and pale yellow colors, respectively. An interactive view is available in the electronic version of the article. (D) Cartoon representation of the superposition of the hFKBD25–FK506 (dark blue) and hFKBD25–rapamycin (pale yellow) with their corresponding inhibitors shown in pale green and grey white stick mode, respectively. Also shown are the two‐dimensional representation of the interactions made by hFKBD25 with FK506 (E) and rapamycin (F). Residues forming hydrogen bonds are shown (labeled in blue) along with those forming nonbonded interactions (labeled in green). It is clearly visible that the hydrogen bonds formed by the backbone oxygen of Gly169 and side‐chain nitrogen of Lys170 with rapamycin (F) are missing in the FK506 complex (E).

Interactions of FK506 with hFKBD25

Molecular interaction between FK506 and hFKBD25 is stabilized by four hydrogen bonds [Fig. 1(C), Table 1] with the backbone atoms of Lys170 and Ile172, and side‐chain oxygen atoms of Asp146 and Tyr198. This is identical to that observed in the hFKBD25–rapamycin and hFKBP12–FK506 complexes [Table 1]. In general, these conserved hydrogen bonds bridges the three ends of FK506 (O2/O10; O3 & O6) with the residues in the active site pocket (Ile172/Lys170; Tyr198 & Asp146), while the pipecolyl moiety forming the fourth end (base of FK506) is mainly stabilized by nonbonded interactions with residues Tyr135, Leu162, Trp175, and Phe216. In addition, many nonbonded interactions, including the C–H…O‐type interactions,11, 12 also stabilize the complex [Fig. 1(C)]. The number of such interactions is higher in the hFKBD25–FK506 complex than those observed in the other two complexes [Table 1].

Table 1.

Interactions Made by FK506 with hFKBD25 and a Comparison with Inhibitor Interactions in hFKBD25–Rapamycin and hFKBP12–FK506

Hydrogen bonds
hFKBD25–FK506			hFKBD25–rapamycin			hFKBD12–FK506
FK506 atom	hFKBD25 atom	Distance (Å)	Rapamycin atom	hFKBD25 atom	Distance (Å)	FK506 atom	hFKBP12 atom	Distance (Å)
O2	Ile172 N	2.8	O2	Ile172 N	2.9	O2	Ile56 N	2.8
O3	Tyr198OH	2.8	O3	Tyr198 OH	2.7	O3	Tyr82 OH	2.8
O6	Asp146OD2	2.6	O6	Asp146 OD2	2.7	O6	Asp37 OD2	2.8
O10	Lys170 O	2.6	O10	Lys170 O	2.7	O10	Glu54 O	2.7
			O8	Lys170 NZ	3.0
			O9	Lys170 NZ	3.0
			O13	Gly169 O	2.9

Nonbonded contacts
hFKBD25–FK506			hFKBD25–rapamycin			hFKBD12–FK506
FK506 atom	hFKBD25 residues		Rapamycin atom	hFKBD25 residues		FK506 atom	hFKBP12 residues
C3	Trp175		C3	Trp175		C3	Trp59
C4	Leu162, Trp175		C4	Leu162, Trp175		C4	Phe46, Val55, Trp59
C5	Tyr135, Leu162, Trp175		C5	Tyr135, Leu162		C12	His87
C6	Tyr135		C43	Ile208		C35	Ile91
C12	Ala206					C36	Tyr26, Phe46
C35	Ala206, Ile208					C41	Phe46
C36	Leu162
C43	Gln203
C45	Tyr198

C–H…O interactions
hFKBD25–FK506			hFKBD25–rapamycin			hFKBD12–FK506
FK506 atom	hFKBD25 residues		Rapamycin atom	hFKBD25 residues		FK506 atom	hFKBP12 residues
O2	Val171, Ile172		O2	Val171, Ile172		O2	Val55, Ile56
O3	Tyr198, Phe216		O3	Tyr198, Phe216		O3	Phe99
O4	Tyr135, Phe145, Asp146, Phe216		O4	Tyr135, Phe145, Phe216		O4	Tyr26, Phe36, Phe99
O6	Asp146		O8	Lys170		O6	Asp37
			O10	Lys170
			O11	Val171
C6	Tyr135		C2	Tyr198		C6	Tyr26
C11	Tyr198		C11	Tyr198		C11	Tyr82
C12	Gln203		C35	Tyr198		C12	His87
C26	Lys170		C37	Lys170		C15	Tyr26, Asp37
C28	Lys170		C39	Gly169, Lys170		C36	Tyr26
C42	Tyr198		C49	Tyr198		C41	Glu54
C43	Gln203		C52	Gly169		C42	Tyr82
C45	Ala197					C45	Ala81

Comparison with hFKBD25–rapamycin and hFKBP12–FK506 complexes

In the previously solved hFKBD25–rapamycin complex,8 three structural features were reported to be accountable for the slightly lower affinity of rapamycin toward hFKBP25 (K _i = 0.9 nM) in comparison to hFKBP12 (K _i = 0.26 nM) and also hypothesized to be the probable reasons for the lower affinity of FK506 (K _i = 200 nM) toward hFKBP25 in comparison to rapamycin. The proposed structural features are (a) the flexibility of the 40s (β3–β4) loop insertion, (b) substitution of the hydrophobic residue Phe46 (hFKBP12) by Leu162 (hFKBP25) in the active site, and (c) substitutions in 50s (β4–α1) loop. Thus, we superimposed the FK506 complex of hFKBD25 on its rapamycin counterpart [Fig. 1(D)] and analyzed the interactions made by the active site residues [Fig. 1(E,F)], focusing mainly the aforementioned regions. In the hFKBP12 active site, three amino acids Tyr26, Asp37, and Arg42, termed as the triad residues, strongly interact with each other mainly through a salt bridge between Asp37 and Arg42. In hFKBD25, this salt bridge is lost due to the substitution of Arg42 by Asn158, while the other two residues are conserved (Tyr26–Tyr135 & Asp37–Asp146). In addition to this, the Asn158 moves away from the other two residues due to the extension of the 40s loop in hFKBD25. This feature which was already observed and reported in the rapamycin complex of hFKBD25 is also corroborated here. Similarly, the substitution of the conserved Phe46 by Leu162 in hFKBP25 also adopts a similar conformation and interacts with the pipecolyl moieties of FK506 and rapamycin, through hydrophobic contacts [Fig. 1(E,F) and Supporting Information, Fig. S1A]. Thus, these two regions could not be the reason for the difference in affinity between these two inhibitors toward hFKBP25. Then while looking into the interactions made by the residues of the 50s loop, the residue Lys170 provided important clues. Its side‐chain nitrogen atom (NZ) makes two hydrogen bonds with the C26 carbonyl and C27 methoxy oxygen atoms of rapamycin [Fig. 1(F) and Table 1]. Interestingly, these atoms are not present in chemical composition of FK506 resulting in the loss of these hydrogen bonds [Fig. 1(E) and Supporting Information, Fig. S1B] though the Lys170 adopt similar orientation in both these structures. In addition, a part of jeffamine, from the crystallization condition, observed between the FK506 molecule and Lys170 probably compensates for the interactions made by rapamycin. Interestingly, in the hFKBP12 structure, this positively charged residue (Lys170) is substituted by a negatively charged Glu54. A superposition of the FK506 and rapamycin complexes of hFKBP12 revealed that the side chain oxygen of Glu54 does not form hydrogen bonds with neither FK506 nor rapamycin. This observation further substantiates the importance of Lys170 in hFKBP25.

Apart from the above differences, the backbone oxygen of the neighboring residue Gly169 also forms a hydrogen bond with O13 of the cyclohexyl ring of rapamycin, but not with FK506 [Fig. 1(E,F) and Table 1; Supporting Information, Fig. S1B]. One reason could be due to the flip of the cyclohexyl ring of FK506 (by ∼77° in comparison to that of rapamycin), away from Gly169 eliminating the possibility of a hydrogen bond. A similar difference in the cyclohexyl ring orientation could also be observed between the rapamycin and FK506 bound structures of hFKBP12.12 Anyhow, in the case of hFKBP12, a larger amino acid (Gln53) replaces the Gly169, probably counterbalancing for the loss of the hydrogen bonding. To further validate the importance of these two residues (Gly169 and Lys170), we compared the identical and nonidentical active site residues of the FK506 complexes of hFKBP12 with hFKBD25 [Supporting Information, Fig. S2]. Of the total 13 active residues, eight of them were identical while five were nonidentical. The nonidentical residues in hFKBP12/hFKBP25 are Phe46/Leu162, Gln53/Gly169, Glu54/Lys170, His87/Gln203, and Ile90/Ala206. As mentioned already, the interactions made by the residue Leu162 with FK506 or rapamycin are almost similar [Supporting Information, Fig. S1A]. The interaction pattern of the residues His87/Gln203 and Ile90/Ala206 also do not vary much [Table 1]. Thus the other two residues Gly169 and Lys170, located in the 50s loop, could be the probable residues responsible for the differences in the affinity for these inhibitors [Supporting Information, Fig. S2]. Anyhow, in FKBP51/52, a glycine (Gly84) replaces the Gln53 of FKBP12, similar to the Gly169 in FKBP25. When comparing the structures of FKBP51 in complex with FK50613 (PDB ID 3O5R) and rapamycin14 (PDB ID 4DRI), we observed that the hydrogen bond interaction by Gly84 with rapamycin is lost in FK506, similar to FKBP25. But the Glu54 in FKBP12, which is substituted by a Gln85/Glu85 in FKBP51/52, does not make hydrogen bonds with rapamycin unlike the equivalent Lys170 in FKBP25, revealing the importance of Lys170, which is highly unique to FKBP25. In conclusion, the structure of hFKBD25 in complex with FK506 and their comparisons presented here have helped us unravel a possible molecular basis for its lower affinity toward hFKBP25 compared to rapamycin, although the role of Lys170 remains to be probed to substantiate our observation.

Materials and Methods

Protein expression and purification

The gene encoding the C‐terminal FK506‐binding domain (FKBD25) of hFKBP25 was amplified by PCR and the PCR amplicon was cloned into a pET29b expression vector. Positive clones were selected by colony PCR and were confirmed by DNA sequencing. For protein expression, clones were transformed into the BL21 (DE3) Escherichia coli strain. Cells were grown till an optical density of 0.6 and then protein expression was induced by 0.5 mM IPTG for 4 h at 25°C. Furthermore, cells were harvested and then lysed by sonication. Finally, the protein was purified by Ni‐NTA chromatography using 20 mM phosphate buffer, 100 mM NaCl, pH 7, and 500 mM Imidazole, which was exchanged to 25 mM Tris buffer pH 7 and 100 mM NaCl and the purified protein was concentrated to 12 mg/mL for crystallization.

Crystallization and X‐ray diffraction experiments

Crystallization screen was performed using the hanging‐drop vapor diffusion method, with hFKBD25 at 12 mg/mL mixed with FK506 at a molar ratio of 1:2 and incubated overnight at 4°C. Equal volumes of the protein and reservoir solutions were mixed and sealed with 500 µL of reservoir solution in each well. Crystals of hFKBD25–FK506 complex appeared in 0.1M HEPES pH 7.0 and 30% v/v Jeffamine ED‐2001 pH 7 after 5–6 weeks at 18°C. The crystals were cryoprotected with 20% glycerol added to reservoir solution for data collection at 100 K on beamline 13B1 at the National Synchrotron Radiation Research Center (Hsinchu, Taiwan) using an ADSC Q315 detector.

Structure determination

The data was indexed, integrated, merged, and scaled using the software iMosflm15 and SCALA16 from CCP4 suite of programs.17 The crystal belonged to the trigonal space group P3₂ 2 1, with one molecule in the asymmetric unit. The initial phases were obtained by molecular replacement calculated using PHASER18 and the protein atoms from the hFKBD25–rapamycin complex (PDB ID 1PBK)8 were used as the search model. REFMAC19 and COOT20 were used for refinement and map fitting respectively while PyMOL21 was used to generate the figures. The electron density for the FK506 atoms could be identified unambiguously at the active site. Additional electron density could be observed at the C‐terminal end corresponding to residues Leu225 and Glu226, resulting from a cloning artifact, followed by two His residues of the 6X His tag. Water molecules were manually picked from the Fo‐Fc and 2Fo‐Fc electron density map contoured at 3.0 and 1.0σ cut‐offs, respectively. In addition, a part of the jeffamine (Ligand Id: 6JZ) could be identified near the active site, while another one, with a few missing atoms, could be located near the β5–β6 loop. The crystallization condition has been the source of this jeffamine. The hFKBD25–FK506 interactions were identified using LigPlot,22 Poseview,23 and manual inspection while the structure based sequence alignment was performed by PROMALS3D24 and EsPript.25 The data collection and refinement statistics are summarized in Table 2.

Table 2.

X‐ray Crystallographic Data Collection and Refinement Statistics for the hFKBD25–FK506 Complex Structure

Data collection
Wavelength (Å)	1.000
Space group	P 32 2 1
Unit cell parameters
a; b; c (Å)	74.78; 74.78; 44.62
α; β; γ (º)	90.00; 90.00; 120.00
Resolution (Å)	30.00–1.83 (1.90–1.83)a
R merge	0.045 (0.556)
Unique reflections	12952 (1264)
Mean [(I)/σ(I)]	34.4 (2.0)
Completeness	99.7 (98.3)
Multiplicity	5.5 (3.2)
Refinement
Number of reflections	11605
Resolution (Å)	25.00–1.83
R‐value	0.1870
R‐Free	0.2331
No. of atoms
Total/protein/FK506/hetero/water	1146/949/57/34/106
Mean B‐value (Å2)
Total/protein/FK506/hetero/water	25.41/23.16/22.56/54.37/37.75
R.m.s.d. from ideal values
Bond lengths (Å)	0.011
Bond angles (º)	1.549
Torsion angles (º)	7.079
Ramachandran statistics (%)
Preferred regions	95.0
Allowed regions	5.0
Outliers	0.0

^aValues in parentheses correspond to those of the highest resolution shell.

Data Deposition

The atomic coordinates and structure factors of the hFKBD25–FK506 complex have been deposited in the Protein Data Bank with accession code 5D75.

Supporting information

Supporting Information