Dynamic NMR studies of ligand‐receptor interactions: Design and analysis of a rapidly exchanging complex of FKBP‐12/FK506 with a 24 kDa calcineurin fragment

Abstract

Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, are normally reserved for the study of small molecule ligand interactions with large protein receptors. Using a combination of isotope labeling and isotope edited NMR, we have extended these techniques to characterize interactions of a much larger protein/drug complex, FKBP-I2/ FK506 with its receptor protein, calcineurin. In order to examine this multicomponent system by dynamic NMR methods, the 93 kDa, tightly bound FKBP-I2/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-I2/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA).Analysis Complexes of the potent immunosuppressants FK506 (Fig. 1) and cyclosporin A with the immunophilins FKBP-12 and cyclophilin, respectively, have been demonstrated to inhibit signal transduction pathways leading to T-cell activation by binding to and inhibiting the protein phosphatase, calcineurin (Cn) (Liu et al., 1991). Since the immunophilin/drug complex and not the drug or immunophilin protein alone is the inhibitory species, information regarding the interaction of this complex with its downstream target, calcineurin, is considered critical for the structure-based design of improved immunosuppressive agents. Unfortunately, the ternary complexes of both FKBP-12IFK506 and Cyp/CSA with calcineurin are both large (>93 kDa) and preclude the use of NMR techniques for a direct structure determination. Therefore, we have focused upon alternative methods to probe the interaction of FKBP-l2/FK506 with calcineurin. The 93 kDa, tightly bound FKBP-l2/FK506/Cn complex was replaced with a lower affinity, rapidly exchanging system consisting of FKBP-12/FK506 (13 kDa), recombinant calcineurin subunit B (CnB) (20 kDa), and a synthetic peptide (4 kDa) corresponding to the B binding domain (BBD) of calcineurin catalytic subunit A (CnA) (Fig. 2) Dynamic NMR methods, such as differential line broadening and transferred NOE spectroscopy, normally reserved for the study of small molecule interactions with large protein receptors (reviewed in Ni, 1994), have been extended to characterize interactions of a much larger proteiddrug complex, FKBP-IUFK506 with a 24 kDa fragment of its receptor protein, calcineurin. Differential line broadening effects and additional NOE contacts for FK506 in the FKBP-I2/FK506/CnB/BBD quaternary complex versus the binary FKBP-l2/FK506 complex have been observed, and indicate which regions of the FK506 molecule are most influenced by interaction with calcineurin.

Kinetic and Structural Characterization of the Effects of Membrane on the Complex of Cytochrome b 5 and Cytochrome c

“…For instance, a rather flexible residue may be rigidified upon binding with cyt c , and its resonance will thus be broadened due to a slower tumbling rate. In addition to correlation time effects, resonance lines may be broadened due to a shift in population levels between different residue conformers 46 .…”

High-field solution NMR spectroscopy as a tool for assessing protein interactions with small molecule ligands

“…By detecting relaxation differences in different regions of the FK506/FKBP complex upon binding to calcineurin, the region of the complex that interacts with the protein target was identified, which was important because the immunophilin/drug complex (FKBP/FK506 respectively), not the drug or protein alone, inhibits an immune response. 55 Differential line broadening assays form the basis of the SHAPES screening method. A small but diverse library known as the SHAPES library is screened against any protein target of interest.…”

Transferred13CT1Relaxation at Natural Isotopic Abundance: A Practical Method for Determining Site-Specific Changes in Ligand Flexibility upon Binding to a Macromolecule

“…13 C T 1 relaxation can provide one of the clearest measurements of atomic-level dynamic behavior of molecules in solution, and it has already served as an important tool for evaluating the internal dynamics of free peptides and inhibitors − and macromolecules. − The T 1 relaxation process has already been described theoretically elsewhere. − Conceptually, the relaxation process occurs via an energy transfer from a 13 C nucleus to its surroundings (lattice) and is highly sensitive to the overall tumbling rate of the molecular system and the local motion of the 13 C nucleus that occur on the pico- to nanosecond time scales. However, the measurement of T 1 data for macromolecule-bound ligands − has severe practical limitations. The low sensitivity and natural isotopic-abundance (1.1%) of the magnetically active 13 C nucleus requires the use of high sample concentrations and the laborious synthesis of isotopically enriched ligands.…”