FKBP12-Rapamycin Complex
Created by Michelle Tran
The protein of interest is the FKBP12-rapamycin complex (PBD ID: 1FAP). It is a ternary complex consisting of two proteins: the FK506-binding protein ( FKBP12) and the human FKBP-rapamycin-associated protein (FRAP), and a ligand: the immunosuppressant agent, rapamycin ( RAP) (Choi, Chen, Schreiber, & Clardy, 1996). FKBP12 is a binding protein that belongs to a family of proteins with prolyl isomerase activity and has been identified in many eukaryotic organisms (Mayer et al., 1997). FRAP, also known as mTOR (mammalian target of rapamycin), belongs to a family of phosphatidylinositol kinase-related kinases involved in controlling the phosphorylation states of translational and cell-cycle regulators, which affect signaling pathways that regulate cell growth, cell proliferation, cell motility, cell survival, protein synthesis, and transcription (Peterson, Beal, Comb, & Schreiber, 2000). FRAP contains a FKBP-rapamycin-binding ( FRB) domain, which is a very important site that interacts with the ligand, RAP (Liang, Choi, & Clardy, 1999). RAP, also known as sirolimus (Wilson et al., 1995), is a bacterially derived macrolide lactone consisting of a 31-membered ring (C56H89NO14) that binds both FKBP12 and FRAP, inhibiting their activity (Leone et al., 2006), therefore becoming a significant and promising therapeutic agent with both immunosuppressant and anti-cancer properties (Law, 2005). It was discovered as a product of the bacterium Streptomyces hygroscopicus in a soil sample from an Easter Island known as "Rapa Nui" and has contributed a great deal to the field of cancer and transplant surgery (Schrier, 2005). RAP has been shown to decrease the frequency of tumor formation after organ transplant by halting tumor cell proliferation, inducing tumor cell apoptosis, suppressing tumor angiogenesis, and also preventing allograft (tissue from a different host) rejection by inhibiting T and B-cell proliferation (Law, 2005).
The FKBP12-rapamycin ternary complex has a roughly rectangular shape with overall dimensions of 60 angstrom by 45 angstrom by 35 angstrom (Choi et al., 1996). The two protein components are virtually the same size, with FKBP12 (blue) having an atomic weight of approximately 12 kDa, and FRAP (red), which has an overall atomic weight of 298 kDa, but has a small, but very important soluble protein FRB domain that is also 12 kDa (Choi et al., 1996). The FRB domain is important because it is where the ligand, RAP, will interact and bind both proteins subunits even more closely together. Although around the same size, these proteins have very different structures. FKBP12 consists of a large beta-sheet composed of five anti-parallel beta-strands (Choi et al., 1996), while FRB consists of a four alpha-helix bundle (alpha1-alpha4) connected by short underhand loops (Liang et al., 1999). Each alpha-helix in the FRB domain is comprised of about 16 to 19 residues (ignoring the first 10 residues of alpha3, the longest helix), but some have unique characteristics, such as alpha3 having a 60 degree bend at Tyr-2074 (black), and alpha2 having a small region, shown in black ( Gly-2049 to Leu-2051) that deviates from a standard alpha helix (Choi et al., 1996). The alpha1 and alpha2 helices are almost parallel to each other at 22 degrees, just like alpha3 and alpha4 at 20 degrees, but the alpha1-alpha2 pair and the alpha3-alpha4 pair crossing angles range from 30 degrees - 60 degrees, which shows that the parallel helices are closer to each other. It is in these interhelical regions that most of the hydrophobic and aromatic residues are located (Choi et al., 1996). Thus, in the last helices, alpha1 and alpha4, a deep cleft is form, which is lined by six aromatic side chains, near the crossing points, allowing it to form a hydrophobic pocket where RAP can bind later on (Choi et al., 1996).
Although part of the same complex, FKBP12 and FRAP have limited protein-protein interactions compared to their extensive RAP-protein interactions (Liang et al., 1999). The main interactions are between the two loops, 40s and 80s, of FKBP12 and the alpha1 and alpha4 of FRAP's FRB (Liang et al., 1999). The interactions between the 40s loop of FKBP12 and the alpha4 helix of FRB are mainly polar hydrogen bonds formed by six water molecules (Choi et al., 1996). Only specific residues in 40s loop of FKBP12, the O atom of residue Lys-47 (black) and the side-chain atoms of Lys-44 (blue) and Phe-46 (orange), have van der Waal contacts with the alpha4 of FRB directly, but there are also other residues, such as Arg-42, Lys-44, Pro-45, and Lys-47 (black) that also contact FRB through water-mediated bridges (Liang et al., 1999). As for the 80s loop of FKBP12, it is connected to the alpha1 of FRB by water-mediated bridges through the NH2 group of Arg-2042 in FRB when it contacts O(gamma) l of Thr85 and the O atom of Gly-86 of FKBP12 (Choi et al., 1996). A special function of the 80s loop is that it changes when in the binary (FKBP12-RAP) complex than when it is in the ternary (FKBP12-RAP-FRB) complex with the major change being around the main and side-chains of residue Ile90 (Choi et al., 1996). The changes in the residue causes FKBP12 to move away from the FRB domain, as if repelled, thus the FRB pocket is optimized for RAP binding (Choi et al., 1996).
There are three subunits; two proteins and one ligand in the complex, therefore, there is a possibility for a binary and ternary complex. The binary complex is the FKBP12 subunit connected with RAP, while the ternary complex is a FKBP12-RAP-FRB complex. RAP cannot bind to FRB as efficiently by itself, but once it is connected with FKBP12 in the binary complex, it is able to bind to FRB tightly (Liang et al., 1999) due to RAP's ability to occupy two different hydrophobic binding pockets (in aquamarine and purple) lined with aromatic residues, simultaneously (Choi et al., 1996). This special characteristic of RAP makes it part of a very limited group of cell-permeable molecules which acquires a gain of function to simultaneously bind two proteins (Liang et al., 1999). The binary complex, FKBP12-RAP, is formed initially when RAP binds to FKBP12 in a hydrophobic pocket that is the result of a short alpha-helix pressed against the beta-sheet (Choi et al., 1996). Three loops in FKBP12 also help contribute to the binding pocket; the 40s loop, which is a bulge in beta5, the 80s which connects alpha2 to the alpha3-sheet, and the 50s loop connecting beta5 to the short alpha-helix (Choi et al., 1996). RAP has a very high affinity to FKBP12 and when bound, inhibits its peptidyl-prolyl isomerase (PPIase) activity (Liang et al., 1999). The immunosuppressive properties of RAP does not have to do with the inhibition of FKBP12's PPIase activity, because PPIase inhibitors are not immunosuppressive agents, rather, immunosuppression is caused by the binary complex's ability to inhibit certain steps in the cytoplasmic signal transductions cascades (Liang et al., 1999). Once formed, the binary complex is able to inhibit FRAP by binding to its FRB domain to form the ternary complex.
The ternary complex, FKBP12-RAP-FRB, is formed when the ligand, RAP, binds to FKBP12, and the composite surface, or the surface with contributions from both RAP and FKBP12, mediates the heterodimerization of FKBP12 and FRAP through the ligand hydrophobic pockets (orange) (Choi et al., 1996). In order to create the complex, RAP must rotate 9 degrees relative to the body of FKBP12, and that results in a change in the FKBP12-binding and FRB-binding domains, which is necessary for the triene region (C17-C24) of RAP to fit into FRB (Liang et al., 1999). Once in the ternary complex, about half of RAP is exposed on the exterior of the complex, and the other half is almost completely buried between the two protein components (Choi et al., 1996). The buried portion of RAP is primarily responsible for the creation of the protein structure because it forms extensive connections with each protein. Just a few of the extensive interactions between RAP and FKBP12 are when the base-forming residue, Trp59, of the hydrophobic binding pocket contacts the pipecolinyl ring (C2-N7 in light green) of RAP (Choi et al., 1996), which is surrounded by conserved aromatic rings of Tyr26, Phe46, Trp59, and Phe99 (Liang et al., 1999). This ring is also the most deeply buried portion of RAP. Another ring, the pyran ring (purple) of RAP, also makes numerous contacts on one end of the FKBP12-binding domain, along with the C10 hydroxyl that forms a hydrogen bond to the Asp37 side chain (Liang et al., 1999). At the other end of the FKBP12 binding domain, the cyclohexyl ring (orange) makes more contacts with FKBP12, with a C40 hydoxyl group forming a hydrogen bond to the main chain O atom of residue Gln53 (Liang et al., 1999). Two hydrogen interactions can be seen between RAP and FKBP12, and another water-bridge is seen between RAP and FRB, specifically the water molecule W331 (Liang et al., 1999). All of these interactions support the view that buried water molecules constitute an integral, noncovalent component of the protein structure (Szep, Park, Boder, Van Duyne, & Saven, 2009) and help to stabilize the ternary complex. Out of the two hydrophobic pockets that RAP binds, the FRB pocket is shallower than the FKBP12's pocket, therefore, the interactions between FRB and RAP are mostly hydrophobic (Liang et al., 1999). Other than the water-bridge that binds RAP and FRB, the triene arm of RAP (C17-C24 in blue) also contact each other closely by projecting into the pocket (Choi et al., 1996) and making 12 contacts with residues Ser2035, Phe2039, Trp2101, Tyr2105 and Phe2108 in black (Liang et al., 1999). The most deeply buried atom of RAP, the methyl group (C45 in purple) attached to C23 (dark blue), makes a total of ten contacts with FRB and fits into the tiny cavity between Leu2031 and Phe2108 (Liang et al., 1999). All these interactions help create the FKBP12-RAP ternary complex that is an essential immunosuppressive drug in medicine, especially in the fields of transplant surgery and oncology.
The primary and tertiary structure that is similar to the RAP component was found to be FK506, otherwise known as tacrolimus (PDB ID: 1FKJ). FK506 (purple) and RAP (blue) are structurally and functionally similar to each other. Both compete for the same binding site on FKBP12. In fact, half of the chemical structure of FK506 is found to be homologous in RAP (Wilson et al., 1995) and both compete for the same binding site on FKBP12. FKBP12 is most known for binding to FK506, due to the fact that FK506 was among the first macrolide immunosuppressants discovered and preceded the discovery of FRAP. FK506 is used widely in treating patients after allograft organ transplants to reduce and prevent transplant rejection, and it is also used for patients suffering from autoimmune disorders (Mayer et al., 1997), which is very similar functionally to RAP. Although they are very similar, there is an important difference between RAP and FK506. The resulting complexes they form with FKBP12 have very different immunosuppressant actions. The RAP-FKBP12 complex blocks T-cell proliferations in response to interleukin-2 (IL-2), which are signaling molecules that are part of the immune system, while the FK506-FKBP12 complex blocks T-cell activation and release of the IL-2 (Schrier, 2005). Another distinction between FK506 and RAP is that RAP mediates the heterodimerization of FKBP12 and FRAP in order to have its immunosuppressant ability, while FK506 inhibits calcineurin, an intercellular Ca2+-dependent phophatase that activates the T-cells of the immune system (Wilson et al., 1995).