Conformational Entropy of FK506 Binding to FKBP12 Determined by Nuclear Magnetic Resonance Relaxation and Molecular Dynamics Simulations

Abstract

FKBP12 (FK506 binding protein 12 kDa) is an important drug target. Nuclear magnetic resonance (NMR) order parameters, describing amplitudes of motion on the pico- to nanosecond time scale, can provide estimates of changes in conformational entropy upon ligand binding. Here we report backbone and methyl-axis order parameters of the apo and FK506-bound forms of FKBP12, based on N andH NMR relaxation. Binding of FK506 to FKBP12 results in localized changes in order parameters, notably for the backbone of residues E54 and I56 and the side chains of I56, I90, and I91, all positioned in the binding site. The order parameters increase slightly upon FK506 binding, indicating an unfavorable entropic contribution to binding of TΔ S = -18 ± 2 kJ/mol at 293 K. Molecular dynamics simulations indicate a change in conformational entropy, associated with all dihedral angles, of TΔ S = -26 ± 9 kJ/mol. Both these values are significant compared to the total entropy of binding determined by isothermal titration calorimetry and referenced to a reactant concentration of 1 mM ( TΔ S = -29 ± 1 kJ/mol). Our results reveal subtle differences in the response to ligand binding compared to that of the previously studied rapamycin-FKBP12 complex, despite the high degree of structural homology between the two complexes and their nearly identical ligand-FKBP12 interactions. These results highlight the delicate dependence of protein dynamics on drug interactions, which goes beyond the view provided by static structures, and reinforce the notion that protein conformational entropy can make important contributions to the free energy of ligand binding.

Leveraging Fungal Calcineurin-Inhibitor Structures, Biophysics and Dynamics to Design Selective and Non-Immunosuppressive FK506 Analogs

“…4). As previously reported [31][32][33][34][35] , hFKBP12 interacts with FK506 with high affinity (K d 2.7 ± 0.5 nM). AfFKBP12 and McFKBP12 also interact tightly with FK506 (K d 4.7 ± 0.6 and 3.0 ± 0.7 nM, respectively).…”

“…To identify biophysical variations between FK506 and APX879 interactions with the human and fungal FKBP12 proteins, we performed ITC assays to establish thermodynamic constants (Table 1 and S4). As previously reported (33)(34)(35)(36)(37), hFKBP12 interacts with FK506 with high affinity (Kd 2.7 ± 0.5 nM). AfFKBP12 and McFKBP12 also interact tightly with FK506 (Kd 4.7 ± 0.6 and 3.0 ± 0.7 nM, respectively).…”

Rotational Dynamics of Proteins from Spin Relaxation Times and Molecular Dynamics Simulations

“…Conformational fluctuations and the overall Brownian tumbling of proteins are experimentally accessible through the spin relaxation times of 15 N and 13 C nuclei measured with nuclear magnetic resonance (NMR) techniques. 1 − 7 The spin relaxation rates have been used to, for example, analyze conformational entropies, 1 , 8 − 11 binding entropies, 1 , 12 resolve sampled structures, 3 − 5 , 13 and validate molecular dynamics (MD) simulations. 14 − 21 These analyses are almost exclusively based on the separation of the internal conformational fluctuations and the overall rotational tumbling.…”

RNA-Protein Interaction Analysis of SARS-CoV-2 5′ and 3′ Untranslated Regions Reveals a Role of Lysosome-Associated Membrane Protein-2a during Viral Infection

“…FKBP4 was also found to interact with the 5′ UTR. It is a member of the immunophilin protein family and binds to immunosuppressants FK506 and rapamycin ( 48 ). Furthermore, Ras GTPase activating protein binding protein 2 (G3BP2), which is key to stress granule formation, was found to interact with the 5′ UTR of SARS-CoV-2.…”