Entropy-driven binding of opioid peptides induces a large domain motion in human dipeptidyl peptidase III Gustavo A. Bezerraa, Elena Dobrovetskyb, Roland Viertlmayra, Aiping Dongb, Alexandra Binterc, Marija Abramic´d, Peter Macherouxc, Sirano Dhe-Paganonb,e, and Karl Grubera,1 aInstitute of Molecular Biosciences, University of Graz, A-8010 Graz, Austria; eDepartment of Physiology and bStructural Genomics Consortium, University of Toronto, Toronto, ON, Canada M5G 1L7; cInstitute of Biochemistry, Graz University of Technology, A-8010 Graz, Austria; and dDivision of Organic Chemistry and Biochemistry, Ruđer Boskovic Institute, 10002 Zagreb, Croatia Edited by William W. Bachovchin, Tufts University School of Medicine, Boston, MA, and accepted by the Editorial Board March 9, 2012 (received for review November 2, 2011) Opioid peptides are involved in various essential physiological of action compared with morphine, spinorphin is an analgesic, processes, most notably nociception. Dipeptidyl peptidase III (DPP potentially useful for pain treatment in morphine-resistant cases III) is one of the most important enkephalin-degrading enzymes (14). This opioid peptide was also shown to be a potent and se- associated with the mammalian pain modulatory system. Here we lective antagonist of the receptor P2X3, which is involved in pain describe the X-ray structures of human DPP III and its complex with signaling in chronic inflammatory nociception and neuropathic the opioid peptide tynorphin, which rationalize the enzyme’s sub- pain due to nerve injury (15). strate specificity and reveal an exceptionally large domain motion Tynorphin (Val-Val-Tyr-Pro-Trp), a synthetic, truncated form upon ligand binding. Microcalorimetric analyses point at an en- of spinorphin, is a highly specific inhibitor of DPP III and was tropy-dominated process, with the release of water molecules shown to induce an even more potent antinociceptive effect from the binding cleft (“entropy reservoir”) as the major thermo- (14, 16). An important role of DPP III in the mammalian pain dynamic driving force. Our results provide the basis for the design modulatory system is supported by several recent findings: low of specific inhibitors that enable the elucidation of the exact role levels of DPP III activity were detected in the cerebrospinal fluid BIOCHEMISTRY of DPP III and the exploration of its potential as a target of pain of individuals suffering from acute pain (17); DPP III exhibits intervention strategies. high in vitro affinity toward the important neuropeptides endo- morphin-1 and endomorphin-2 (18); and neuroanatomical stud- isothermal titration calorimetry | metallopeptidase | peptide binding | ies localized rat DPP III in the superficial laminae of the dorsal X-ray crystallography horn, where enkephalin-synthesizing neurons and high concen- trations of endomorphin-2 are found (19, 20). he endogenous opioid system, composed of opioid peptides DPP III is also related to other physiologic processes. It is Tand their receptors, modulates a large number of physiological overexpressed in ovarian primary carcinomas, the aggressiveness processes, such as endocrine and immune function, gastrointesti- of which is correlated with enhanced DPP III activity (21). In a nal motility, respiration, reward, stress, complex social behavior recent genomic screen, it was also identified as a potential acti- (e.g., sexual activity), vulnerability to drug addiction, and most vator of the antioxidant response element by inducing nuclear notably the procession and transmission of pain stimuli (noci- translocation of NF-E2–related factor 2 (22). Recently, DPP III ception) (1, 2). Two major types of endogenous opioid peptides was found among the approximately 1,100 proteins that constitute are those containing enkephalin sequences at the N terminus (Tyr- the human central proteome, which is the set of proteins ubiq- Gly-Gly-Phe-Met/Leu) (3) and, more recently identified, endo- uitously and abundantly expressed in all human cell lines (23). morphins 1 and 2 (Tyr-Pro-Trp/Phe-Phe-NH2) (4, 5). Knowledge Advances in pain therapy have been scarce in the past decades. and control over synthesis and degradation pathways of this class Acute and chronic pain are disabling conditions affecting millions of molecules is prerequisite for the development of new therapies of people worldwide, with huge social costs associated (24). Novel that target pertinent physiological processes. pharmacological compounds such as enkephalin-degrading en- Dipeptidyl peptidase III (DPP III), also known as enkephalinase zyme inhibitors were shown to possess analgesic properties while B, is an enkephalin-degrading enzyme that cleaves dipeptides se- lacking the adverse effects of some current therapeutics agents, quentially from the N termini of substrates (6). All DPP IIIs de- such as morphine and its surrogates (25). However, degradation scribed thus far contain the unique zinc-binding motif HEXXGH of enkephalins is effected by the joint action of several enzymes characteristic of metallopeptidase family M49 (7). Enzymes from [i.e., NEP, aminopeptidase N (APN), and DPP III] (26), and several human and animal tissues, as well as from lower eukar- inhibiting only one of those enzymes yields just marginal effects. yotes, were purified and biochemically characterized (8, 9). DPP For instance, RB101, a dual inhibitor of APN and NEP, was able III is largely found as a cytosolic protein, although membrane association in rat brain and Drosophila melanogaster has been de- scribed (10, 11). The 3D structure of the yeast ortholog has re- Author contributions: G.A.B., M.A., P.M., S.D.-P., and K.G. designed research; G.A.B., E.D., cently been determined, revealing a unique protein fold with two R.V., A.D., and A.B. performed research; G.A.B., A.D., M.A., P.M., S.D.-P., and K.G. ana- lyzed data; and G.A.B., M.A., P.M., S.D.-P., and K.G. wrote the paper. lobes forming a wide-open substrate-binding cleft (12). The lack of fl structural information on peptide complexes, however, left the The authors declare no con ict of interest. question of substrate specificity largely unanswered. This article is a PNAS Direct Submission. W.W.B. is a guest editor invited by the Editorial Board. DPP III purified from monkey brain microsomes is strongly Data deposition: Crystallography, atomic coordinates, and structure factors reported inhibited by the neuropeptide spinorphin (Leu-Val-Val-Tyr-Pro- in this paper have been deposited in the Protein Data Bank, www.pdb.org (PDB ID codes Trp-Thr), an endogenous factor isolated from bovine spinal cord 3FVY, 3T6B, and 3T6J). that also inhibits other enkephalin-degrading enzymes, such as 1To whom correspondence should be addressed. E-mail: [email protected]. neutral endopeptidase (NEP, neprilysin), aminopeptidase, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. angiotensin-converting enzyme (13). Because of a different mode 1073/pnas.1118005109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1118005109 PNAS Early Edition | 1of6 Downloaded by guest on September 27, 2021 to increase enkephalin levels and to produce antinociceptive, an rmsd of 1.4 Å for 523 superimposed Cα-atoms (SI Appendix, antidepressant, and antianxiety effects in rodents without opioid- Fig. S2). related side effects, but the potency of this compound was much For cocrystallizing hDPP31–726 with the opioid peptide tynor- lower compared with morphine (27). phin, we exchanged Glu-451 for Ala, rendering the enzyme in- To date, DPP III is the only known enkephalin-degrading en- active (29) and preventing peptide cleavage. Two different, zyme for which no structural data on substrate or inhibitor com- monoclinic crystal forms were obtained under the same con- plexes are available. This lack of information has been a major ditions: space group P21 with two molecules per asymmetric unit obstacle for a detailed understanding of the enzyme’s substrate and space group C2 with one molecule per asymmetric unit. specificity and for the development of specific inhibitors. There- These crystals diffracted to 2.4 and 3.0 Å resolution, respectively. fore, we determined the crystal structure of human DPP III and Structure solution by molecular replacement failed when we its complex with the opioid peptide tynorphin, revealing a large used the complete structure of the unbound enzyme. However, domain movement upon ligand binding, which possesses the when the two lobes were used separately as search templates, signature of a distinctly entropy-driven process. a solution was obtained (SI Appendix, Methods). In both structures we observed well-defined electron density for Results and Discussion tynorphin bound between the two lobes (SI Appendix, Fig. S3). Initial attempts to crystallize the full-length human DPP III yiel- The two independent molecules in the 2.4-Å structure are almost ded crystals unsuitable for X-ray structure determination. To identical to each other (Cα-rmsd 0.03 Å) and—despite the dif- improve crystal quality a truncated form was designed (called here ferent packing environments in the two crystal forms—they are α hDPP31–726), lacking 11 amino acid residues at the C terminus also very similar to the 3.0-Å structure (C -rmsd of 0.5 Å). Be- that were predicted to be unstructured (28). Monoclinic crystals cause of the closely similar overall structures and conformations (space group P21) obtained for this construct yielded diffraction of the bound peptide (SI Appendix, Fig. S4), we are focusing our data up to 1.9 Å resolution. The structure was solved by molecular discussion on the higher-resolution structure. The most striking replacement using the structure of yeast DPP III (12), resulting in difference between unbound hDPP31–726 and its peptide complex one molecule in the asymmetric unit. is the closure of the binding cleft, which completely buries the The overall fold of hDPP31–726 (Fig. 1A) is very similar to the bound ligand (Fig. 1). According to the program DynDom (30, yeast homolog (36% sequence identity).