Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III

Abstract

Opioid peptides are involved in various essential physiological processes, most notably nociception. Dipeptidyl peptidase III (DPP III) is one of the most important enkephalin-degrading enzymes associated with the mammalian pain modulatory system. Here we describe the X-ray structures of human DPP III and its complex with the opioid peptide tynorphin, which rationalize the enzyme's substrate specificity and reveal an exceptionally large domain motion upon ligand binding. Microcalorimetric analyses point at an entropy-dominated process, with the release of water molecules from the binding cleft ("entropy reservoir") as the major thermodynamic driving force. Our results provide the basis for the design of specific inhibitors that enable the elucidation of the exact role of DPP III and the exploration of its potential as a target of pain intervention strategies.

Fig. 1.

Overall structure of human DPP III. (A) Two perpendicular views of the overall structure of unbound hDPP3_1–726. The upper, zinc-binding lobe (residues 337–374 and 422–668) is shown in light blue, and the lower lobe (residues 1–336, 375–421, and 669–726) is shown in pink. The five-stranded β-core in the lower lobe (residues 307–335 and 376–409) is highlighted in magenta; the zinc ion is depicted as a yellow sphere. (B) Two perpendicular views of the overall structure of the complex with the opioid peptide tynorphin.

Fig. 2.

Structure of human DPP III in complex with the opioid peptide tynorphin. (A) Close-up view of the five stranded β-core in the lower lobe (magenta), which is extended by the bound peptide (yellow). (B) Superposition of the zinc-binding residues in the structures of the unbound enzyme (light blue) and the peptide complex (pink). The peptide is shown in yellow, and the zinc ion is represented as a gray sphere. The supposed interaction of the P1 carbonyl group with the zinc ion is shown as a magenta dashed line. The interaction of a water molecule (W) with the zinc ion (see also SI Appendix, Fig. S1) as well as hydrogen bonds are depicted as green dashed lines. (C) Polar interactions of tynorphin with human DPP III. Residues from the lower lobe are shown in pink, His-568 (from the upper lobe) in light blue, and the peptide in yellow.

Fig. 3.

Domain movement upon tynorphin binding to human DPP III. (A) Surface representation of unbound hDPP3_1–726 (Left) and of the peptide complex (Right). The coloring scheme is identical to that in Fig. 1. (B) Close-up view of the hinge region (residues 409–420) shown in orange. Lys-670 is shown in blue, together with its polar interactions with the U-shaped part of the hinge region. (C) Superposition of the hinge regions and Lys-670 in the unbound structure (orange) and in the peptide complex (cyan).

Fig. 4.

Microcalorimetric analysis. (A) ITC measurement at 15 °C. Upper: Time-dependent deflection of heat for each injection. Lower: Peak integral as a function of molar ratio of hDPP3_1–726 to tynorphin. The continuous curve represents the best fit using a one-site binding model. (B) Table with thermodynamic data derived from the ITC measurements at different temperatures. (C) Temperature dependence of ΔG, ΔH, and -TΔS. (D) Surface representation of unbound hDPP3_1–726, indicating water molecules bound in the cleft and the part of the surface buried upon complex formation (light blue).