Structure-based classification of 45 FK506-binding proteins

Abstract

The FK506-binding proteins (FKBPs) are a unique group of chaperones found in a wide variety of organisms. They perform a number of cellular functions including protein folding, regulation of cytokines, transport of steroid receptor complexes, nucleic acid binding, histone assembly, and modulation of apoptosis. These functions are mediated by specific domains that adopt distinct tertiary conformations. Using the Threading/ASSEmbly/Refinement (TASSER) approach, tertiary structures were predicted for a total of 45 FKBPs in 23 species. These models were compared with previously characterized FKBP solution structures and the predicted structures were employed to identify groups of homologous proteins. The resulting classification may be utilized to infer functional roles of newly discovered FKBPs. The three-dimensional conformations revealed that this family may have undergone several modifications throughout evolution, including loss of N- and C-terminal regions, duplication of FKBP domains as well as insertions of entire functional motifs. Docking simulations suggest that additional sequence segments outside FKBP domains may modulate the binding affinity of FKBPs to immunosuppressive drugs. The docking models also indicate the presence of a helix-loop-helix (HLH) region within a subset of FKBPs, which may be responsible for the interaction between this group of proteins and nucleic acids.

Figure 1. FKBP models closely represent previously determined crystal structures

Pairwise structural alignments using ClustalW_3D between empirically generated protein structures and in silico derived models revealed that the TASSER set of programs creates complete protein representations that match closely the known crystal structures. TASSER models are on the top, with the PDB structures at the bottom of each pair. The number bar at the top indicates position of amino acids. A. TASSER generated model of FKBP1 from H. sapiens matches exactly to that of its corresponding conformation in the PDB (PDB ID: 1FKK), with the exception of a single Met in the PDB structure. B. Both N- and C-terminal regions of FKBP5 from H. sapiens are missing in the crystallograph (dashes) compared to the TASSER model. The structural alignment delineates 4 additional internal regions that are incomplete in the PDB conformation. C. Although the N- and C-termini of the E. coli FKBP from TASSER is more complete, the first 102 amino acids are shifted by 1 to 4 positions compared to the PDB structure.

Figure 2. FKBP tertiary structures

Tertiary structures were predicted using the TASSER set of programs and assembled into groups of potential homologues. Models were rendered using PyMol. Figures 1A through 1I correspond to Homo sapiens FKBP1, FKBP2, FKBP3, FKBP4/5, FKBP6, FKBP7, FKBP8, FKBP9/10 and FKBP11/14, respectively. FKBPs from other organisms were placed into groups of orthologues based on structural similarity to one or more of the Homo sapiens FKBPs.

Figure 3. Superposition of Homo sapiens FKBP1 with insect homologues

Putative orthologues were superimposed in a pairwise fashion using the STRAP program to assess the degree of structural similarity among groups of FKBPs. FKBP1 from Homo sapiens (red) is superimposed with FKBPs from (A) Anopheles gambiae (yellow) and (B) Manduca sexta (green). Although FKBPs exist in all known organisms, their cellular roles have not been identified in a number of taxonomic groups. Structural similarity suggests that functional parallelisms may exist in a variety of organisms and the superpositions enable us to hypothesize about the utility of the FKBPs in species for which no function has been characterized.

Figure 4. Docking simulations suggest that FKBP orthologues may have differential affinity for FK506

FK506-FKBP interactions were modeled using the ZDOCK server and compared with previously determined FK506-FKBP solution structures. Docking simulations using Homo sapiens FKBP1 and FK506 correctly predicted the drug active site; however, ZDOCK showed FK506 bound more internally (A) within the active site when compared to the solution structure complex (B). These models revealed the potential for different orthologues to bind FK506 in slightly different orientations, which may reflect subtle differences in affinity among FKBPs.

Figure 5. FKBP45 from Bombyx mori may possess an overlapping active site for both FK506 and RNA

Identification of the potential active site for FK506 (A) and stem-loop RNA (B) within Bombyx mori FKBP45 indicates that the protein may associate with the drug and RNA in the same region. The characteristic FK506- and rapamycin-binding domain is outlined in white brackets. These simulations also suggest that additional domains surrounding the FKBP drug binding pocket may reduce the protein’s affinity for FK506 and/or create novel sites for drug interactions.