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.isothermal titration calorimetry | metallopeptidase | peptide binding | X-ray crystallography T he endogenous opioid system, composed of opioid peptides and their receptors, modulates a large number of physiological processes, such as endocrine and immune function, gastrointestinal motility, respiration, reward, stress, complex social behavior (e.g., sexual activity), vulnerability to drug addiction, and most notably the procession and transmission of pain stimuli (nociception) (1, 2). Two major types of endogenous opioid peptides are those containing enkephalin sequences at the N terminus (TyrGly-Gly-Phe-Met/Leu) (3) and, more recently identified, endomorphins 1 and 2 (Tyr-Pro-Trp/Phe-Phe-NH 2 ) (4, 5). Knowledge and control over synthesis and degradation pathways of this class of molecules is prerequisite for the development of new therapies that target pertinent physiological processes.Dipeptidyl peptidase III (DPP III), also known as enkephalinase B, is an enkephalin-degrading enzyme that cleaves dipeptides sequentially from the N termini of substrates (6). All DPP IIIs described thus far contain the unique zinc-binding motif HEXXGH characteristic of metallopeptidase family M49 (7). Enzymes from several human and animal tissues, as well as from lower eukaryotes, were purified and biochemically characterized (8, 9). DPP III is largely found as a cytosolic protein, although membrane association in rat brain and Drosophila melanogaster has been described (10, 11). The 3D structure of the yeast ortholog has recently been determined, revealing a unique protein fold with two lobes forming a wide-open substrate-binding cleft (12). The lack of structural information on peptide complexes, however, left the question of substrate specificity largely unanswered.DPP III purified from monkey brain microsomes is strongly inhibited by the neuropeptide spinorphin (Leu-Val-Val-Tyr-ProTrp-Thr), an endogenous factor isolated from bovine spinal cord that also inhibits other enkephalin-degrading enzymes, such as neutral endopeptidase (NEP, neprilysin), aminopeptidase, and angiotensin-converting enzyme (13). Because of a different mode of action compared with morphine, spinorp...

Novel dipeptidyl hydroxamic acids that inhibit human and bacterial dipeptidyl peptidase III

“…Further experimental data are required to be able to speculate whether the same enzymic functional group (with pK a 7.5) is influencing binding of hydroxamate inhibitors to hDPP III. We showed recently that the tetrahedral coordination of the catalytic zinc ion (two His, one Glu and one water molecule as ligands) in DPP III closely resembles that observed in metalloendopeptidase thermolysin 17,18 , which has been thoroughly investigated. An equivalent catalytic mechanism has been proposed for DPP III accordingly that involves general base (Glu451 in hDPP III) activation of the zinc-bound water molecule 17 .…”

“…Specificity nomenclature for peptidase interactions with substrate/inhibitor is presented in Supplementary Figure 1. Recently, Bezerra et al 18 resolved the crystal structure of human DPP III in complex with pentapeptide tynorphin (ValVal-Tyr-Pro-Trp), revealing the formation of closed enzyme's active site during the ligand binding which induces a large domain motion. This work defined the amino acid residues forming five deep subsites (S2-S3 0 ) which accommodate the amino acid side chains in the positions P2-P3 0 of the peptide substrate.…”

“…Dipeptidyl hydroxamic acids could be considered as substrate analogs which occupy the S2 and S1 subsite of DPP III. The S2 subsite is in human enzyme formed by Glu316, Ile390, Asn391, Ile392, Asn394, Asp396, Arg399, His450, Trp495, Asp496, Ser504, Glu507 and Glu508 18 . Nine amino acid residues constitute the S1 subsite: Tyr318, Glu329, Phe381, Pro387, Gly389, Ile390, His450, Glu508 and His568.…”

“…We have cloned and biochemically characterized the first bacterial DPP III, from the human gut symbiont B. thetaiotaomicron, revealing similarities but also important differences compared to its human counterpart 16 . Until now, crystal structures of two M49 family members have been solved, yeast and human DPP III 17,18 . The 3-D structure of human DPP III inactive mutant in complex with pentapeptide tynorphin 18 revealed the formation of closed enzyme's active site during the substrate binding and exposed the amino acid residues which form five deep subsites (S2-S3 0 ).…”

“…Until now, crystal structures of two M49 family members have been solved, yeast and human DPP III 17,18 . The 3-D structure of human DPP III inactive mutant in complex with pentapeptide tynorphin 18 revealed the formation of closed enzyme's active site during the substrate binding and exposed the amino acid residues which form five deep subsites (S2-S3 0 ). Due to the low homology (less than 25% sequence identity) to eukaryotic proteins, not one 3-D structure of prokaryotic member of the M49 family has been resolved yet.…”