¹⁵N NMR Relaxation Studies of Backbone Dynamics in Free and Steroid-Bound Δ⁵-3-Ketosteroid Isomerase from Pseudomonas testosteroni

Abstract

The backbone dynamics of Delta(5)-3-ketosteroid isomerase (KSI) from Pseudomonas testosteroni has been studied in free enzyme and its complex with a steroid ligand, 19-nortestosterone hemisuccinate (19-NTHS), by (15)N relaxation measurements. The relaxation data were analyzed using the model-free formalism to extract the model-free parameters (S(2), tau(e), and R(ex)) and the overall rotational correlation time (tau(m)). The rotational correlation times were 19.23 +/- 0.08 and 17.08 +/- 0.07 ns with the diffusion anisotropies (D( parallel)/D( perpendicular)) of 1.26 +/- 0.03 and 1.25 +/- 0.03 for the free and steroid-bound KSI, respectively. The binding of 19-NTHS to free KSI causes a slight increase in the order parameters (S(2)) for a number of residues, which are located mainly in helix A1 and strand B4. However, the majority of the residues exhibit reduced order parameters upon ligand binding. In particular, strands B3, B5, and B6, which have most of the residues involved in the dimer interaction, have the reduced order parameters in the steroid-bound KSI, indicating the increased high-frequency (pico- to nanosecond) motions in the intersubunit region of this homodimeric enzyme. Our results differ from those of previous studies on the backbone dynamics of monomeric proteins, in which high-frequency internal motions are typically restricted upon ligand binding.

Configurational Entropy in Protein–Peptide Binding:

“…Such observations are contrary to the perhaps intuitive expectation that adding a ligand will simply tighten down the protein and produce a global reduction in configurational entropy. However, they are consistent with NMR studies of proteins showing that ligand-binding can tighten the binding site while making other regions of the protein more mobile [10, 11, 14, 64, 65, 66, 67, 68, 69]. In fact, it has been proposed that there is a general “conformational relay” mechanism that balances increased rigidity of some residues against increased flexibility of their neighbors [14].…”

Induction of Flexibility through Protein-Protein Interactions

“…In these cases, binding is associated with a loss of conformational entropy that is necessarily offset by increases in solvent entropy and/or the formation of favorable enthalpic interactions. Decreases/increases in backbone dynamics that are compensated for by increases/decreases (respectively) in distal regions in the backbone, as we report here, have also been found for protein hydrophobic target interactions (21,41,45,(51)(52)(53)(54)(55). It may be that the observed increase in flexibility in the binding pocket accompanies the release of structured water molecules from the solvent accessible hydrophobic surfaces.…”

Increase in Backbone Mobility of the VTS1p-SAM Domain on Binding to SRE–RNA

“…There is increasing evidence from dynamics studies involving binding of peptides, proteins, and singlestranded DNA and RNA to proteins, to indicate that binding does not always lead to motional restriction. 14,15,[36][37][38][39][40] In many of the above examples, increased mobility in much of the protein has been reported with complex pattern of motions across the interface. In single-stranded DNA binding to the Cterminal DNA binding domain of Escherichia coli topoisomerase I, a majority of residues show a slight decrease in S 2 and almost all residues have increased R ex following DNA binding.…”