Calcineurin—immunosuppressor complexes

770

Calcineurin-immunosuppressor complexes Barry L Stoddard* and Karen E Flickt Crystal structures of the Ser/Thr phosphatase calcineurin (protein phosphatase 2B) have recently been solved by X-ray crystallography, both in the free-protein state, and complexed with the immunophilin/immunosuppressant FKBP1 2/FK506. Core elements of the calcineurin phosphatase have been found to be similar to the corresponding elements of Ser/-rhr phosphatase 1 and purple acid phosphatase. The structures provide a basis for understanding calcineurin inhibition by a ternary complex of immunophilin and immunosuppressant proteins.

Addresses Fred Hutchinson Cancer Research Center, 1124 Columbia Street, Seattle, Washin9ton 98104-2092, USA *e-mail: [email protected] re-mail: [email protected] Current Opinion in Structural Biology 1996, 6:770-775 © Current Biology Ltd ISSN 0959-440X Abbreviations CaN calcineurin CsA cyclosporinA IL interleukin KBPAP kidney bean PAP PAP purpleacid phosphatase PP protein phosphatase

Introduction

Immunosuppressive agents such as cyclosporin A (CsA), FK506 and rapamycin are natural microbial products that are probably synthesized as chemical defense agents by their source organism [1]. T h e modes of action of CsA and FK506 have been particularly well studied and have been shown to proceed through the binding of these molecules to specific cytosolic-rcceptor proteins (cyclophilin and FKBP12, respectively) [2-4]. Both of these proteins are small, ubiquitous cis-trans peptidyl-prolyl isomerases that are strongly inhibited upon the binding of their cognate ligands. The inhibition of the isomerasc activities of these enzymes is not sufficient to block immune response [5,6]; rather, these immunophilin-immunosuppressant complexes (cyclophilin-CsA or FKBP12-FK506) suppress T-cell activation and proliferation by binding to the Ser/Thr phosphatase 2B, also known as calcineurin (CAN) [7-9]. Inhibition of calcineurin activity by the formation of these ternary complexes inhibits a number of T-cell specific responses, including activation of specific transcription factors (such as NF-AT, the nuclear factor of activated T cells, and NF-IL2A, a nuclear factor that binds the IL-2 enhancer) [10-13], leading to reductions in the transcription of a number of early genes in the T-cell activation process, such as interleukin (IL)-2, IL-3, IL-4, GMC-SF (granulocyte-macrophage colony-stimulating factor), and

interferon-y. The mode of association of immunophilin, immunosuppressant, and target (calcineurin) is therefore of enormous interest for the design of novel immunosuppressant compounds (as well as immunoactivators). Calcineurin is strongly activated by both calcium and calmodulin as part of its role in the T-cell activation pathway. T h e identity of the natural target of calcineurin in T cells is under intense study, with in vitro studies suggesting at least one lymphocyte-specific nuclear transcription factor, NF-ATp [13]. This protein is dephosphorvlated and translocated to the nucleus following intracellular calcium release, where it stimulates expression of the IL-2 gene. As a member of the Ser/Thr phosphatase family of enzymes, calcineurin (PP2B) displays a high degree of sequence homology with other members of the same enzyme family; including PPl and PP2A [14,15]. Members of this family of enzymes have highly homologous catalytic domains, but differ in their substrate specificities and interactions with regulatory molecules (Fig. 1). T h e entire collection of Ser/Thr phosphatases is unrelated in sequence and architecture to the tyrosine phosphatases. Full-length calcineurin is a heterodimer composed of an A subunit (CaNA, 59 kDa) and a B subunit (CaNB, 19kDa). A variety of studies have identified four distinct functional domains of CaNA: a catalytic domain, a CaNB-binding domain, a calmodulin-binding domain, and an autoinhibitory domain [16-18]. CaNB binds four calcium ions and has 35% sequence identity with calmodulin. T h e phosphatase activity of CaNA is stimulated both by calcium binding to CaNB, and by calcium-induced binding of calmodulin to CaNA [19]. Structural

studies

T h e X-ray crystallographic structures of several variants of calcineurin and of the complex of calcineurin with FKBP12-FK506 have been reported in the past year by two separate laboratories [20**,21"]. During the past two years, the structure of the mammalian PPA, the kidney bean purple acid phosphatasc (KBPAP) and human PP1 were also reported [22*'-24°°], allowing the direct comparison of several members of the Ser/Thr phosphatase-enzyme families. Statistics relevant to these structures are summarized in Table 1. T h e polypeptide chains of the inhibited-calcineurin complex (CaNA, CaNB and FKBP12) form a roughly rectangular structure, with the largest component being CaNA of the phosphatase. T h e core of the phosphatasc domain is formed by a [3-sandwich motif similar to the core of DNAse I [25] and cxonuclease III [26]. CaNA presents a long 22-residue helix that extends nearly 40 1~ from the surface of the phosphatase domain. This helix forms the primary binding site for CaNB, which associates with one side of the helix using a hvdrophobic groove

Calcineurin-immunosuppressor complexes Stoddard and Flick 771

Figure 1

320 0

Protein phosphatase 1 25

293

~Regulatory Phosphorylation

Protein phosphatase 2A 414

67

348

482

3!

4

Protein phosphatase 2B (Calcineurin A subunit) CaNB Binding

CaM AutoBinding inhibitory

Inserts

(2.5 A resolution complex) v

(2.1 A resolution free protein, 3.5 A complex)

Schematic diagram showing domain structure and regulatory regions of Ser/'rhr phosphatases. The residue numbers given refer to protein phosphatase (PP)I from rabbit muscle, human PP2A, and the A subunit (CaNA) of human calcineurin (PP2B). The site of regulatory phosphorylation in PP1 and the site of B subunit (CaNB) binding, calmodulin (CAM) binding, and the autoinhibitory domain of CaNA are shown. The relative length of the calcineurin constructs used for crystallographic studies is indicated in the lower part of the diagram by arrows for bovine (shorter arrow, [20"°]) and human (longer arrow, [21"]) calcineurin. Adapted with permission from [2].

Table 1 Summary of structural information for calcineurin, related phosphatases and inhibitors. Structure

Source

Resolution (A)

R-factor

Reference

CaNA-CaNB + FKBP12-FK506"

Cow

2.5

0.220

[20"]

Full-length CaNA-CaNB (free protein)

Human

2.1

0.186

[21 °°]

Full-length CaNA-CaNB + FKBP12-FK506

Human

3.5

0.262

[21 "°]

Protein phosphatase-1

Rabbit

2.1

0.178

[22"]

Purple acid phosphatase

Kidney bean

2.9

0.196

[23"]

Protein phosphatase-1A

Human

2.5

0.201

[24"]

FKBP12-rapamycin-FRAP complex

Human

2.7

O.193

[36 °°]

*CaNA truncated before CaM domain. CaM, calmodulin; CaNA, A subunit of CaN; CaNB, B subunit of CaN; FRAP, FKBP-rapamycin-associated protein.

that is formed by a pair of calmodulin-like domains. T h e opposite side of the helix, which is completely exposed in the heterodimetic free enzyme, is the binding site for the FKBP12-FKS06 complex. T h e active site of the enzyme (which is found in the conserved catalytic domain

of CaNA) is located more than IOA away from any other component of the ternary complex. T h e overall architecture of C a N A [20",21 °°] is shown in Figure 2. As described above, the core of this structure

772

Catalysis and regulation

is a [3-sandwich motif, and contains the enzyme active site. Located above the closed end of the 13 sandwich, the active-site residues are found within four loops and at the C-terminal ends of two [3 strands, forming a shallow pocket that is quite wide and should allow access by macromolecular phosphorylated substrates (such as transcription factors) for dcphosphorylation. T h e active site contains two metal ion binding sites (for ZnZ+ and Fe 3+ ions), in positions that arc consistent with those in the catalytic core of purple acid phosphatase (PAP), which possesses sequence and structure similar to calcineurin in this region [23"']. T h e ions arc identified on the basis of their respective ligands and geometries (one aspartate, one asparagine, two histidine residues coordinate ZnZ+; two aspartate, one histidine and one water coordinate Fe 3+. Both metals have a coordinating oxygen from a bound phosphate that is missing in the PAP structure) [20°',21°',23"°]. T h e metal ions are located 3/~, apart in the active site, and have an aspartate side chain that acts as a monodcntate-bridging ligand between the metals. T h e bound phosphate in the active site could represent the labile phosphate group required during dephosphorylation of the substrate. It is stabilized by interactions with guanidinium groups of two argininc residues and with the Ne2 of a single histidine residue. T h e phosphate oxygen coordinated to this histidinc residue could represent the leaving oxygen of the substrate, on the basis of its location opposite the Fe3+-bound water molecule, with the histidine serving as a proton donor to the leaving group in an SN2-type mechanism. Based on the high level of sequence and structure conservation between the catalytic domains of all Ser/Thr phosphatases of the PP1, PP2A, and PP2B subfamilies, one can speculate on a plausible mechanism for the activity of calcineurin and related Ser/Thr protein phosphatases. A watcr or hydroxyl group, acting as a bridging ligand between zinc and iron, would be within 2.9.;~ of the modeled phosphorus, and directly in line with the P - O scissile bond of the substrate. This ligand could act as a nucleophile and attack the phosphorus, leading to a pentacoordinate transition-state structure that would be stabilized by contacts with the conserved arginine and histidine residues in the active site. Calcineurin's phosphatase activity, is stimulated by calmodulin binding to CaNA, and also by Ca 2+ binding to CaNB of the heterodimer. Although the structures of the phosphatase alone or in complex with immunophilin/immunosuppressor do not appear to suggest a mechanism by which this activation occurs, the binding of CaNB is by itself quite striking. CaNB, consisting of two sequential globular CaZ+-binding domains, is associated in a linear manner along the long amphipathic ot helix of CaNA described above. T h e two domains of CaNB can be roughly superimposed by a translation of 22/~ along the helix. This arrangement is quite distinct from the manner by which calmodulin binds to cz helices, in which the two domains are on opposite sides of the helix and related by a twofold rotation axis [27]. It is hypothesized by one

Figure 2

Ribbon diagram of the active A subunit (CaNA) of human calcineurin (PP2B). The Fea+ and Zn 2+ ions (small spheres) are bound in a cleft in the catalytic domain. The two central ~ sheets form a distorted 13sandwich, with sheet 1 (upper) consisting of six J5 strands and sheet 2 (lower) consisting of five ~ strands. The surface of sheet 1 is covered by three (z helices and a three-stranded 13 sheet, whereas sheet 2 is covered by an all-o~-helical structure. The active site is located at the closed end of the ~ sandwich, and includes the two metal ions and a bound phosphate ion (not shown). Figure generated using RIBBONS [39] and used with permission from James Griffiths (Vertex Pharmaceuticals, Cambridge, MA, USA).

group [21 °°] that the CaZ+-bound lobes of CaNB serve to anchor the two subunits of the enzyme and transduce the structural and dynamic effects of CaZ+ binding to the catalytic domain. Of perhaps the greatest importance of these structures for potential biomedical applications and for structurebased drug design is the description of the association and inhibition by the immunophilin-immunosuppressant complex (FKBP12-FK506) [20",21"*]. T h e conformation of the binding protein FKBP12 is nearly identical to that found in the structure of the FKBP1Z-FK506 binary

Calcineurin-immunosuppressor complexes Stoddard and Flick

complex [28-30]. T h e conformations of the immunosuppressant FK506 are also virtually identical among these structures. T h e entire complex binds to calcineurin along the extended (x helix, opposite CaNB, making contact with the helix, CaNB, and the catalytic domain of CaNA. Approximately 400 to .550 A2 of solvent-accessible surface area is buried for each component upon formation of the complex; several of the residues forming the protein interface have been identified on the basis of site-directed mutagenesis experiments as participating in the inhibition of calcineurin activity [31-33]. As mentioned above, the bound immunosuppressor complex is located over 10~ from the active site, and, therefore, does not directly interfere with the chemical mechanism of dephosphorylation; rather it is likely that the bound complex interferes with the association of calcineurin with the necessary protein substrates for T-cell 'activation. T h e structures of full-length human calcineurin [21°°], both in the absence of bound immunosuppressor complex and when bound to the complex, add significant additional details to the basic pattern of protein association described above. T h e CaN heterodimer alone is found to be identical in conformation to the complexed enzyme, including the presence of the extended helix which forms the binding region for CaNB. There are three significant contributions of this structural study. First the N-terminal segment of CaNA in the human enzyme make an important contribution to the CaNB-binding interface by forming part of a binding cleft for the C-terminal lobe of CaNB. Second, an 18 residue segment of the C-terminal region of CaNA (residues 469 to 486) lies over the apparent substrate-binding cleft in the catalytic domain of the free enzyme, accounting for the at, toinhibito U properties of that region of the protein (Fig. 1). These residues form two short c~ helices and the residues that interact with the catalytic region are the most highly conserved residues within the autoinhibitory segment. The observed binding of this C-terminal region is consistent with two reports of competitive inhibition of calcineurin by a 25-residue peptide consisting of the same sequence [34,35]. Third, the immunosuppressor complex FKBP12-FK506 induces the displacement of the autoinhibitory peptide from the shallow active-site pocket of the enzyme. Finally, a recent crystallographic structure has been reported for the FKBP12 immunophilin protein bound with a different immunosuppressant molecule (rapamycin) and complexed to a macromolecular target (FKBP-rapamycinassociated protein, or FRAP) that is quite distinct from calcineurin [36"]. Rapamycin, when complexed with FKBP12, effects cell-cycle behavior through a mechanism that interrupts signals from the IL-2 receptor and the receptors for other cytokines and growth factors. T h e features of this structure support the previous structural work on FKBP in complex with FKS06 (both irnmunosuppressant agents bind to the same site on FKB12),

773

and add detail to our knowledge of the process of immunosuppression. Conclusions

As is routine in many crystallographic experiments, the structure of the calcineurin-immunosuppressor complex, while enormously revealing in detail about the pattern of protein recognition and association for this system, leaves several open questions regarding the mechanism of enzyme activation and inhibition by Ca2+ and FKBP12-FK506, respectively. Although the most obvious conclusion for the mechanism of inhibition is simple steric hindrance of substrate binding, there are features of complex formation and its kinetic effect on dephosphorylation that are difficult to explain on the basis of the existing structures. One feature is the classical noncompetitive inhibition of calcineurin by the immunosuppressor complex [23"], which implies the formation of a catalytically inactive enzyme-substrate-inhibitor complex, with no part of the active site or substrate binding cleft directly obstructed. A second feature is the measurable kinetic inhibition of calcineurin-dependent dephosphorylation activity in the presence of bound immunophilin-immunosupressor complexes [37], and a third is the observation that a small 22 amino acid peptide encompassing the FKBPlZ-homology region of a different inhibitor protein (AKAP79) also acts as noncompetitive inhibitor of calcineurin [38]. This peptide would appear, on the basis of homology modeling, to provide minimal steric hindrance to substrate binding. These open questions will need to be answered by further biochemical and structural investigations. T h e inhibition and activation of calcineurin is a key step in the T-cell activation pathway and therefore is of enormous interest for the design of immt,nosuppressan[ compounds. Studies of the activity and inhibition of calcincurin will continue, and hopefully the future will provide significant progress toward the application of this knowledge to biomedical uses and the treatment of human disease.

References and recommended reading Papers of particular interest, published within the annual period of review, have been highlighted as: • •*

of special interest of outstanding interest Williams DH, Stone MJ, Hauck PR, Rahman SK: Why are secondary metabolites (natural products) biosynthesized? J Nat Prod 1989, 52:1189-1208.

8ierer BE, Mattila PS, Standaert RF, Herzenberg LA, Burakoff S J, Crabtree G, Schreiber SL: Two distinct signal transmission pathways in T lymphocytes are inhibited by complexes formed between an immunophilin and either FK506 or rapamycin. Proc Nat/Acad Sci USA 1990, 87:9231-9235. Handschumacher RE, Harding MW, Rice J, Drugge RJ: Cyclophilin: a specific cytosolic binding protein for cyclosporin A. Science 1984, 226:544-547.

7?4

Catalysis and regulation

Tropschug M, Barthelmess IB, Neupert W: Sensitivity to cyslosporin A is mediated by cyelophilin in Neurospora crassa and Saccharomyces cerevisiae. Nature 1989, 342:953-955. Heitman J, Vovva NR, Hiestand PC, Hall MN: The FK506 ° binding protein proline rotamase is a target for the immunosuppressive agent FK506 in Saccharomyces cerevisiae. Prec Natl Acad Sci USA 1991, 88:1948-1952. Koltin Y, Faucette L, Bergsma DJ, Levy MA, Cafferkey R, Koser PL, Johnson RK, Levi GP: Rapamycin sensitivity in Saccharomyces cerevisiae is mediated by a peptidyl-prolyl cis-trans isomerase related to human FK506-binding protein, Mol Cell Bio/1991, 11:1718-1723. Liu J, Farmer JD Jr, Lane WS, Friedman J, Weissman I, Schreiber SL: Calcineurin is a common target of cyclophilin-cyclosporin A and FKBP-FK506 complexes. Cell 1991, 66:807-815. O'Keefe S J, Tamura J, Kincaid RL, Tocci MJ and O'Neill EA: FK506 and CsA-sensitive activation of the interleukin-2 promoter by calcineurin. Nature 1992, 357:692-694. Clipstone NA and Crabtree GR: ldentificetion of calcineurin as a key signaling enzyme in T-lymphocyte activation. Nature 1992, 357:695-697. 10.

Mattila PS, Ulman KS, Fiering S, Emmel EA, McCutcheon M, Crabtree GR, Herzenber9 LA: The actions of cyclosporin A and FKS06 suggest a novel step in the activation of T lymphocytes. EMBO J 1990, 9:4425-4433.

11.

Emmel EA, Verweij CL, Durand DB, Higgins KM, Lacy E, Crabtree G: Cyclosporin A specifically inhibits function of nuclear proteins involved in f cell activation. Science 1989, 246:1617-1620.

12.

Randak C, Brabletz T, Hergenrother M, Sobotta I, Serfling E: Cyclosporin A suppresses the expression of the interleukin 2 gene by inhibiting the binding of lymphocyte-specific factors to the IL-2 enhancer. EMBO J 1990, 9:2529-2536.

13.

Flanagan WM, Corthesy B, Bram PJ, Crabtree GR: Nuclear association of a T-cell transcription factor blocked by FK-506 and cyclosporin A. Nature 1991, 352:803-807.

14.

Ito A, Hashimoto T, Hirai M, Takeda T, Shuntoh H, Kuno 1", Tanaka C: The complete primary structure of calcineurin A, a calrnodulin binding protein homologous with protein phosphatases 1 and 2A. Biochem Biophys Res Commun 1989, 163:1492-1497.

15.

Cohen Prw: Two isoforms of protein phosphatase 1 may be produced from the same gene, FEBS Lett 1988, 232:17-23.

16.

Klee CB, Crouch TH and Krinks MH: Calcineurin: a calcium- and calmodulin-binding protein of the nervous system. Proc Nat/ Acad Sci USA 1979, 76:6270-6273.

1"7.

Hashimoto Y, Perrino BA, Soderling TR: Identification of an autoinhibitory domain in calcineurin. J Biol Chem 1990, 265:1924-1927.

t8.

19.

20. oo

Watanabe Y, Perrino BA, Chang BH, Soderling TR: Identification in the calcineurin A subunit of the domain that binds the regulatory B subuniL J Blot Chem 1995, 270:456-460.

activity of calcineurin by physically hindering the approach of protein substrates to the active site. 21. •.

Kissinger CR, Parge HE, Knighton DR, Lewis CT, Pelletier LA, TempczykA, Kalish VJ, Tucker KD, Showalter RE, Moomaw EW et aL: Crystal structures of human calcineurin and the human FKBP12-FK506-calcineurin complex. Nature 1995, 378:641-644. The X-ray structures of full-length human calcineurin at 2.1/~ resolution and the human calcineurin complex with FKBP12/FK506 at 3.5/~ resolution are presented. Some additional details regarding the structural role of the autoinhibitory domain and the N-terminal region of the protein are provided by the complex structure. The authors hypothesize that the inhibitory properties of the immunophilin-immunosuppressant complex may be due to long-distance dynamic or structural effects imposed on the active site residues of the enzyme, in addition to indirect physical hindrance of substrate binding. 22. •.

Goldberg J, Huang H-B, Kwon Y-G, Greengard P, Nairn AC, KunyanJ: Three-dimensional structure of the catalytic subunit of protein serine/threonine phosphatase-l. Nature 1995, 376:745-753. The X-ray structures of mammalian protein phosphatase 1 at 2.1/~ resolution, complexed with the toxin microcystin, is presented. The fold of the catalytic domain and the architecture of the active-site side-chain network {s ctosely preserved in calcineurin. 23. •-

Strater N, Klabunde T, Tucker P, Witzel H, Krebs B: Crystal structure of a purple acid phosphatase containing a dinuclear Fe (lll)-Zn (11) active site. Science 1995, 268:1489-1492. The first structure of a bimetallic Ser/Thr protein phosphatase is reported at 2.9~ resolution. The coordination and identity of the metal-ion cofactors serve as the initial model for the active sites of calcineurin reported in [20 "°] and [21°']. The authors' hypothesis of a mechanism for phosphate ester hydrolysis involving nucleophilic attack by an iron-coordinated hydroxide ion is supported by the subsequent structural studies of PP1 and calcineurin. 24. •.

EgloffMP, Cohen PTW, Reinemer P, Barford D: Crystal structure of the catalytic subunit of human protein phosphatase 1 and its complex with tungstate. J Mo/Bio/1995, 254:942-959. The structure of PPI-A in its active conformation is presented. The structure was solved on the based of muttiwavelength anomalous diffraction phasing from a tungstate molecule bound at the catalytic site. The nature of the metal was definitively established by protein-induced X-ray emission spectroscopy. 25.

Oefner C, Suck D: Crystallographic refinement and structure of DNase I at 2A resolution. J Mo/Bio11986, 192:605-632.

26.

Mol CD, Kuo CF, Thayer MM, Cunningham RP, Tainer JA: Structure and function of the multifunctional DNA-repair enzyme exonuclease III. Nature 1995, 374:381-386.

27.

Meador WE, Means AR, Quiocho RA: Target enzyme recognition by calmodulin: 2.4 A structure of a calmodulin-peptide complex. Science 1992, 257:1251-1255.

28.

Van Duyne GD, Standaert RF, Karplus PA, Schreiber SL, Clardy J: Atomic structure of FKBP-FK506, an immunophilin-immunosuppressant complex. Science 1991, 252:839-842.

29.

Becker JW, Rotonda J, McKeever BM, Chan HK, Marcy AI, Wiederrecht G, Hermes JD, Springer JP: FK-506-binding protein: three-dimensional structure of the complex with the antagonist L-685,818. J Bio/Chem 1993, 268:t 1335-1 t 339.

30.

Wilson KP, Yamashita MM, Sintchak MD, Rotstein SH, Murcko MA, Boger J, Thomson JA, Fitzgibbon MJ, Black JR, Navia MA: Comparative X-ray structures of the major binding protein for the immunosuppressant FK506 (tacrolimus) in unliganded form and in complex with FK506 and rapamycin. Acta Crystallogr D 1995, 51:511- 521.

31.

Aldape RA, Futer O, DeCenzo MT, Jarrett BP, Murcko MA, Livingston DJ: Charged surface residues of FKBP12 participate in formation of the FKBP12-FK506-calcineurin complex. J Biol Chem 1992, 267:16029-16032.

32.

Yang D, Rosen M K and Schreiber SL: A composite FKBP12-FK506 surface that contacts calcineurin. J Am Chem Soc 1993, 115:819-820.

Stemmer PM, Klee CB: Dual calcium ion regulation of calcineurin by calmodulin and calcineurin B. Biochemistry 1994, 33:6859-6866.

GriffithJP, Kim JL, Kim EE, Sintchak MD, Thomson JA, Fitzbiggon MJ, Fleming MA, Caron PR, Hsiao K, Navia MA: X-ray structure of calcineurin inhibited by the immunophilin-immunosuppressant FKBP12-FK506 complex. Cell 1995, 82:507-522. The X-ray structure of the ternary complex of a bovine calcineurin CaNA fragment (missing the CaM binding region and autoinhibitory region), CaNB, FKBP12, and the immunosuppressant drug FK506 at 2.5/~ resolution is presented. The authors hypothesize that binding of the immunophilin-immunosuppressant complex inhibits the dephosphorylation

Calcineurin-immunosuppressor complexes Stoddard and Flick

33.

Futer O, DeCenzo MT, Aldape RA, Livingston DJ: FKS06 binding protein mutational analysis. J Biol Chem 1995, 270:18935-18940.

34.

Parsons JN, Wiederrecht G J, Salowe S, Kincaid RL, O'Keefe SJ: Regulation of calcineurin phosphatase activity and interactions with the FKS06-FK506-binding protein complex. J Biol Chem 1994, 269:19610-19616.

35.

36. •.

Lewis C, Gastinel L, Habuka N, Tucker K, Chen X, Maldonado F, Villafranca JE: Characterization of human recombinant calcineurin heterodimer co-expressed in bacteria and insect cells [abstract]. FASEB J 1995, 9:1346. Choi J, Chen J, Schreiber SL, Clardy SL: Structure of the FKBP12-rapamycin complex interacting with the binding domain of human FRAP Science 1996, 273:239-242.

??5

The crystal structure of the ternary complex of human FKBP12, rapamycin, and the FKBP-rapamycin bindin£ domain of human FRAP (FKBP-rapamycin-associated protein) at 2.7A resolution reveals the two proteins are bound together as a result of the ability of rapamycin to occupy two different hydrophobic binding pockets simultaneously. 37.

Swanson SK, Born T, Zydowsky LD, Cho H, Chang HY, Walsh CT, Rusnak F: Cyclosporin-mediated inhibition of bovine calcineurin by cyclophilins A and B. Proc Nail Acad Sci USA 1992, 89:3741-3745.

38.

Coghlan VM, Perrino BA, Howard M, Langeberg LK, Hicks JB, Gallatin WM, Scott JD: Association of protein kinase A and protein phosphatease 2B with a common anchoring protein. Science 1995, 267:108-111.

39.

Carson M: RIBBONS 2.0../Appl Crystallogr 1991, 24:958-961.