Entropy-driven binding of 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 , 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 -degrading enzymes (14). This opioid 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 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 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 endo- morphin-1 and -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 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 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 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, ), aminopeptidase, and This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. BIOCHEMISTRY -converting enzyme (13). Because of a different mode 1073/pnas.1118005109/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1118005109 PNAS | April 24, 2012 | vol. 109 | no. 17 | 6525–6530 Downloaded by guest on September 29, 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). A wide cleft separates 31), this conformational change can be described by a movement a mostly helical upper lobe containing the conserved zinc-bind- of two rigid bodies corresponding to the two lobes of the protein. ing motifs (450-HELLGH-455 and 507-EECRAE-512; zinc-co- This movement involves a rotation of approximately 60° of one ordinating residues in boldface in SI Appendix, Fig. S1) from a domain relative to the other, with negligible translational con- lower lobe of α/β-secondary structures containing a five-stranded tributions. A superposition on the corresponding upper and lower β-barrel core (12). A superposition of the two structures yielded domains in the bound and unbound structures yielded Cα-rmsd values between 0.4 and 0.6 Å, indicating the rigidity of these domains. A difference distance matrix calculated from the Cα- coordinates of the bound and unbound structures revealed the same picture, with the upper and lower lobes moving as rigid bodies (SI Appendix, Fig. S5). Tynorphin is bound in an extended conformation. The first three residues of the peptide form a β-strand, which binds to the five-stranded β-core of DPP III in an antiparallel fashion (Fig. 2A). Such a binding mode involving the extension of a β-sheet by the ligand is not uncommon in protein–peptide complexes (32). In both complex structures, electron density was missing for the zinc ion, which was very likely lost during crystallization. A superposition of the zinc-containing structure of unbound hDPP31–726 onto the structure of the peptide complex, however, reveals that the conformations of the zinc-coordinating residues are conserved in both structures (Fig. 2B). In this superposition, the carbonyl oxygen of the scissile peptide bond (P1, Val-2) is close to the zinc-bound water molecule (present in the structure of the unbound enzyme; SI Appendix, Fig. S1) and is at a distance of approximately 2.7 Å from the catalytic ion itself. Similarly, the side chain of Glu-451 (of the unbound protein) is appropriately positioned to activate this water molecule for the nucleophilic attack onto the amide bond. As in the proposed mechanisms of thermolysin (33) and neprilysin (34), the hydrogen bond between His-568 and the carbonyl group of Val-2 (Fig. 2B) likely provides additional stabilization of the tetrahedral intermediate (SI Ap- pendix, Fig. S6). Both Glu-451 and His-568 are completely conserved among known DPP IIIs (9, 35). The N terminus of the bound peptide is completely buried by the enzyme and anchored by polar interactions to the side chains Fig. 1. Overall structure of human DPP III. (A) Two perpendicular views of of Glu-316 and Asn-394 and to the main-chain carbonyl group of the overall structure of unbound hDPP31–726. The upper, zinc-binding lobe Asn-391 (Fig. 2C). These tight interactions provide a rationale (residues 337–374 and 422–668) is shown in light blue, and the lower lobe fi (residues 1–336, 375–421, and 669–726) is shown in pink. The five-stranded for the observation that peptides with modi ed N termini are β – – not accepted as substrates by DPP III (36). Additional hydrogen -core in the lower lobe (residues 307 335 and 376 409) is highlighted in fi β magenta; the zinc ion is depicted as a yellow sphere. (B) Two perpendicular bonds to the ve-stranded -core as well as to Tyr-318 suggest views of the overall structure of the complex with the opioid peptide that the correct positioning of the P1 carbonyl group close to the tynorphin. catalytic zinc ion is the dominant basis of the dipeptidyl

6526 | www.pnas.org/cgi/doi/10.1073/pnas.1118005109 Bezerra et al. Downloaded by guest on September 29, 2021 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 ex- tended 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 car- bonyl 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 hy- drogen 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.

peptidase specificity of this enzyme. This is supported by the fact binding, this lysine residue moves away to interact with the C- that all four of these catalytic residues are conserved among DPP terminal tryptophan, leading to a conformational change in the IIIs (9, 35) and that a 125-fold decrease of kcat/Km was observed hinge region (Fig. 3C). Interestingly, Lys-670 is also highly con- in the Y318F variant of human DPP III (37). served. In the yeast enzyme this position is occupied by an arginine Substrate specificity of peptidases is likely governed by the that forms similar interactions with the corresponding part of the shape and properties of the corresponding subsites in the binding hinge region (SI Appendix,Fig.S8). site. In DPP IV and DPP VII, for example, subsite S1 is small The secondary structure at the beginning of the hinge is largely and hydrophobic, restricting P1 to proline, alanine, or glycine preserved upon ligand binding and conformational change (Fig. (38). In contrast, all subsites in DPP III are deep and somewhat 3). The majority of the hinge residues have hydrophobic side hydrophobic (SI Appendix, Fig. S7 and Table S2), in agreement chains, and therefore primarily weak interactions have to be with the relaxed specificity shown by this enzyme (18). rearranged upon domain motion, mainly as a result of side-chain The C-terminal carboxylate of the bound peptide forms a salt rotations. In addition, the temperature factors of these residues bridge with Arg-669 (Fig. 2C). This residue is one of a series of are not significantly different from the average B-factors in the conserved, positively charged amino acids (“Arg anchors”) in the rest of the protein. These observations suggest that the hinge binding cleft that have previously been predicted to bind the C motion is a low-energy deformation similar to the situation first termini of different-length peptides (12). Although the N termi- observed in T4 lysozyme (40). nus of the bound peptide is completely shielded from solvent, the In the complex structure, tynorphin forms polar interactions cleft is more spacious on the opposite side, and a narrow tunnel almost exclusively with residues of the lower lobe (Fig. 2C). The connects the cleft with the exterior (SI Appendix, Fig. S7). This only exception is the hydrogen bond between His-568 and the structural feature partly explains the observation that DPP III is carbonyl group of residue P1, which is important for catalysis (SI able to accommodate peptides up to a length of 10 residues (39). Appendix, Fig. S6). In addition, the larger part of the surface of The analysis of the domain movement upon peptide binding the bound peptide is buried through interactions with the lower using the program DynDom (30, 31) yielded a mainly rotational lobe. On the basis of this pattern of interactions, we suggest an motion of approximately 60° and identified amino acids 409–420 order of peptide binding to human DPP III: the peptide binds as “mechanical hinge” residues (Fig. 3). The magnitude of this first to the lower lobe (especially by interactions with five- conformational change in enzymes is uncommon, probably be- stranded β-core; Fig. 2); the C terminus of the bound peptide cause of the entropic costs involved (32). induces conformational changes in the hinge region (Fig. 3C), Residues in the hinge region are evolutionarily conserved possibly triggering the observed domain motion; and the latter among DPP IIIs, particularly at the beginning of the stretch, where brings the catalytic zinc ion (over a distance of approximately they form secondary structures (9, 35). The C-terminal part of this 16 Å) in close juxtaposition to the carbonyl group of the scissile

hinge region adopts a distinct U-shaped conformation that is amide bond. A possible role of substrate binding in triggering the BIOCHEMISTRY stabilized by hydrogen bonds to Lys-670 (Fig. 3B). With tynorphin conformational change is corroborated by the observation that

Bezerra et al. PNAS | April 24, 2012 | vol. 109 | no. 17 | 6527 Downloaded by guest on September 29, 2021 Fig. 3. Domain movement upon tynorphin binding to human DPP III. (A) Surface representation of unbound hDPP31–726 (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).

a minimum number of four residues are required for a peptide To further characterize tynorphin binding, we performed a series being efficiently cleaved by DPP III (36, 41, 42). Furthermore, of isothermal titration calorimetry experiments at four different the close similarity of the unbound, open structures of human temperatures ranging from 5 to 35 °C. In this temperature range and yeast DPP III, despite their moderate sequence identity (SI peptide binding was strongly endothermic (Fig. 4A), although Appendix, Fig. S2), indicates that this open structure represents exothermic binding was expected on the basis of the supposition adefined, even fixed conformation of the protein rather than that peptide interactions with proteins are mainly governed by a random snapshot from a continuum of flexible, open optimization of hydrogen bonds (32). To have a spontaneous pro- conformations. cess, the free energy (ΔG), at constant pressure, must be negative, Because the bound peptide is completely buried between the and hence the entropy term has to outweigh the enthalpy term (i.e., lobes and only narrow channels connect the binding site with the TΔS > ΔH) (43) in an endothermic process. As shown in Fig. 4, the bulk solvent (SI Appendix, Fig. S7), product release quite likely positive ΔH in the entire temperature range studied is counter- occurs after reopening of the cleft. acted by a large gain in entropy, resulting in an exergonic binding

6528 | www.pnas.org/cgi/doi/10.1073/pnas.1118005109 Bezerra et al. Downloaded by guest on September 29, 2021 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 hDPP31–726 to tynorphin. The continuous curve represents the best fit using a one-site binding model. (B) Table with thermo- dynamic data derived from the ITC measurements at different temperatures. (C) Temperature dependence of ΔG, ΔH, and -TΔS.(D) Surface representation of

unbound hDPP31–726, indicating water molecules bound in the cleft and the part of the surface buried upon complex formation (light blue).

process. The enthalpy term is less unfavorable at higher temper- consider the water molecules bound in the large cleft between atures and overcompensates the smaller contribution by the en- the two lobes as an “entropy reservoir” used to drive the binding tropy term, resulting in almost 40-fold tighter binding at 35 °C process. The endothermic binding behavior of endomorphin-1, compared with 5 °C. Overall, the temperature dependence of the Leu-enkephalin, and IVYPW (SI Appendix, Fig. S9 and Table Δ enthalpy (heat capacity change, Cp)hasanegativeslope,in- S3) indicates that the same concept very likely applies more dicating that the binding process is dominated by the burial of generally to the binding of other opioid peptides to DPP III. hydrophobic surfaces. An analysis of the buried surface area upon DPP III is one of very few peptidases known to display an en- peptide binding [using the program NACCESS (44)] indeed yiel- tropy-driven binding mode (49) and/or a large domain movement ded a large loss of solvent-accessible area of approximately 3,500 2 upon substrate binding (50). The latter was suggested to regulate Å , with a ratio between hydrophobic and hydrophilic areas of the enzyme in a mechanical rather than chemical fashion, with Δ fi 60:40. The calculated Cp using empirical area coef cients (45) is the rate-determining step being the closure of the active site (50). − −1 −1 2.5 kJ K mol , in close agreement with the experimentally It is also conceivable that the closure of the binding cleft con- − −1 −1 determined value of 2.6 kJ K mol (Fig. 4C). tributes to the promiscuous substrate specificity of DPP III. The Thus, binding of tynorphin to human DPP III clearly is an domain movement may be seen as a way to accommodate diverse entropy-driven process, with the major contribution very likely substrates through “conformational plasticity” (51) (i.e., by clos- being the release of ordered water molecules from the binding ing differently around different peptides). Along the same lines, cleft of the unbound enzyme (Fig. 4). A layer of water molecules (≈7 Å deep) consisting of different shells with varying rigidity is the release of a large number of water molecules from the binding thought to be present on the surface of protein molecules, and cleft (Fig. 4D) and the concomitant entropy gain facilitate the the shells are in dynamic equilibrium with each other and the binding of different peptides, almost irrespective of the exact bulk solvent (46, 47). In an analysis of crystallization processes, (enthalpic) interaction between enzyme and substrate. the entropy contribution of the release of structured water The insights gained by our structure determination and the molecules has been estimated to be in the range of 100 to 700 J thermodynamic characterization of the binding process also − − mol 1 K 1, corresponding to approximately 5–30 water mole- provide a starting point for the rational design of molecular tools cules displaced upon the incorporation of a protein molecule into specific for DPP III and pave the way for new efforts to exploit a crystal (48). In the ligand-free structure of DPP III approxi- this enzyme as drug target for pain intervention strategies. DPP mately 60 water molecules are positioned in the binding cleft III inhibitors might offer alternatives to conventional treatments (Fig. 4D), and we assume that the displacement of all (or most in the nociceptive field, especially the use of of) these waters is sufficient to explain the favorable entropy agonists—such as morphine and its derivatives—that possess se- −1 −1 contribution to ligand binding (from 170 to 450 J mol K in vere side effects. The data also allow the design of inhibitors that BIOCHEMISTRY the temperature range of our measurements). Therefore, we do not target the zinc ion but rather interfere with the domain

Bezerra et al. PNAS | April 24, 2012 | vol. 109 | no. 17 | 6529 Downloaded by guest on September 29, 2021 motion. This would decrease cross-reactivity with other metal- against the ratio of peptide vs. protein concentration in the cell. Nonlinear lopeptidases and thus unwanted side effects. least-squares fitting to these binding isotherms was used to obtain association constants (Ka), heats of binding (ΔH), and stoichiometries. Kd and Gibbs free Methods energy (ΔG) were calculated. A truncated variant of human dipeptidyl peptidase III consisting of residues ACKNOWLEDGMENTS. We thank Lissete Crombet for cloning services; Alma 1–726 (hDPP31–726) was expressed in SF9 cells, purified to homogeneity by column chromatography, and crystallized. For cocrystallization with the opi- Seitova for generating recombinant baculovirus; Denise Cavalcante Hissa and Bernhard Lehofer for assistance in protein purification; Christian C. Gruber oid peptide tynorphin an inactive variant (E451A) of the enzyme expressed in for assistance in generating the distance difference matrix; and the beamline Escherichia coli was used. Diffraction data to 1.9 Å (unbound enzyme) as well staff at the Advanced Photon Source, the Deutsches Elektronensynchrotron/ as to 2.4 Å and 3.0 Å (complexes with tynorphin) were collected using syn- European Molecular Biology Laboratory, and the European Synchrotron chrotron radiation, and the structures were solved by molecular replacement Radiation Facility for support during diffraction data collection. Funding was using the structure of the yeast homolog as template (12). provided by Project W901 (Doktoratskolleg “Molecular Enzymology”) from Isothermal titration calorimetry (ITC) data were carried out at various the Austrian Science Funds; the Austrian Agency for International Coopera- temperatures in 25 mM Tris·HCl (pH 7.5) and 200 mM NaCl. Binding of the tion in Research and Education; Project 098-1191344-2938 from the Croatian Ministry of Science, Education and Sport; the Canadian Institutes for Health peptides (tynorphin, endomorphin-1, Leu-enkephalin, and IVYPW) to hDPP3 – 1 Research; the Canadian Foundation for Innovation; Genome Canada through 726 E451A was analyzed with a VP-ITC microcalorimeter (MicroCal) equilibrated μ the Ontario Genomics Institute; GlaxoSmithKline; Karolinska Institutet; the at the respective temperature. A 200- M peptide solution in the syringe was Knut and Alice Wallenberg Foundation; the Ontario Innovation Trust; the μ titrated into a 40- M solution of the enzyme in the measuring cell. The cor- Ontario Ministry for Research and Innovation; Merck & Co., Inc.; the Novartis responding heats of binding were determined by integration of the observed Research Foundation; the Swedish Agency for Innovation Systems; the Swed- peaks after correction for the heat of dilution of the peptide and plotted ish Foundation for Strategic Research; and the Wellcome Trust.

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