The structural basis of Edc3- and Scd6-mediated activation of the Dcp1:Dcp2 mRNA decapping complex

The Dcp1:Dcp2 decapping complex catalyses the removal of the mRNA 5' cap structure. Activator proteins, including Edc3 (enhancer of decapping 3), modulate its activity. Here, we solved the structure of the yeast Edc3 LSm domain in complex with a short helical leucine-rich motif (HLM) from Dcp2. The motif interacts with the monomeric Edc3 LSm domain in an unprecedented manner and recognizes a noncanonical binding surface. Based on the structure, we identified additional HLMs in the disordered C-terminal extension of Dcp2 that can interact with Edc3. Moreover, the LSm domain of the Edc3-related protein Scd6 competes with Edc3 for the interaction with these HLMs. We show that both Edc3 and Scd6 stimulate decapping in vitro, presumably by preventing the Dcp1:Dcp2 complex from adopting an inactive conformation. In addition, we show that the C-terminal HLMs in Dcp2 are necessary for the localization of the Dcp1:Dcp2 decapping complex to P-bodies in vivo. Unexpectedly, in contrast to yeast, in metazoans the HLM is found in Dcp1, suggesting that details underlying the regulation of mRNA decapping changed throughout evolution.     

Insights into the Structure of Invisible Conformations of Large Methyl Group Labeled Molecular Machines from High Pressure NMR

Most proteins are highly flexible and can adopt conformations that deviate from the energetically most favorable ground state. Structural information on these lowly populated, alternative conformations is often lacking, despite the functional importance of these states. Here, we study the pathway by which the Dcp1:Dcp2 mRNA decapping complex exchanges between an autoinhibited closed and an open conformation. We make use of methyl Carr–Purcell–Meiboom–Gill (CPMG) NMR relaxation dispersion (RD) experiments that report on the population of the sparsely populated open conformation as well as on the exchange rate between the two conformations. To obtain volumetric information on the open conformation as well as on the transition state structure we made use of RD measurements at elevated pressures. We found that the open Dcp1:Dcp2 conformation has a lower molecular volume than the closed conformation and that the transition state is close in volume to the closed state. In the presence of ATP the volume change upon opening of the complex increases and the volume of the transition state lies in-between the volumes of the closed and open state. These findings show that ATP has an effect on the volume changes that are associated with the opening-closing pathway of the complex. Our results highlight the strength of pressure dependent NMR methods to obtain insights into structural features of protein conformations that are not directly observable. As our work makes use of methyl groups as NMR probes we conclude that the applied methodology is also applicable to high molecular weight complexes.     

Conformational effects of C(alpha,alpha)-dipropargylglycine as a constrained residue

A useful synthon to approach artificial phenylalanyl peptides in a [2 + 2 + 2] cycloaddition reaction, C(alpha,alpha)-dipropargylglycine (Dprg) is examined for its conformational preferences as a constrained residue. Crystal structure analysis and preliminary NMR results establish possible preference of the residue for folded (alpha) rather than extended (beta) region of the straight phi,psi conformational space. Boc-Dprg-L-Leu-OMe (1) displays two molecular conformations within the same crystallographic asymmetric unit, with Dprg in the alpha(R) or alpha(L) conformation, participating in a type I beta-turn or an alpha(L)-alpha(R)-type fold, in which Leu(2) assumes the alpha(R) conformation stereochemically favored for an L-chiral residue. Boc-Dprg-D-Val-L-Leu-OMe (2) displays a type I' beta-turn conformation in crystal, with both Dprg(1) and D-Val(2) assuming the alpha(L) conformation stereochemically favored for a D-chiral residue, with 4 --> 1 type hydrogen bond linking L-Leu(3) NH with Boc CO. NMR analysis using temperature variation, solvent titration, and a spin probe study suggests a fully solvent-exposed nature of Dprg NH, ruling out a fully extended C(5)-type conformation for this residue, and solvent sequestered nature of L-Leu(3) NH, suggesting possibility of a beta-turn due to Dprg assuming a folded conformation.     

Structural basis for HNF-4alpha activation by ligand and coactivator binding

In addition to suggesting that fatty acids are endogenous ligands, our recent crystal structure of HNF-4alpha showed an unusual degree of structural flexibility in the AF-2 domain (helix alpha12). Although every molecule contained a fatty acid within its ligand binding domain, one molecule in each homodimer was in an open inactive conformation with alpha12 fully extended and colinear with alpha10. By contrast, the second molecule in each homodimer was in a closed conformation with alpha12 folded against the body of the domain in what is widely considered to be the active state. This indicates that although ligand binding is necessary, it is not sufficient to induce an activating structural transition in HNF-4alpha as is commonly suggested to occur for nuclear receptors. To further assess potential mechanisms of activation, we have solved a structure of human HNF-4alpha bound to both fatty acid ligand and a coactivator sequence derived from SRC-1. The mode of coactivator binding is similar to that observed for other nuclear receptors, and in this case, all of the molecules adopt the closed active conformation. We conclude that for HNF-4alpha, coactivator rather than ligand binding locks the active conformation.     

The crystal structure of Afc-containing peptides

A systematic structural analysis of Afc (9-amino-fluorene-9-carboxylic acid) containing peptides is here reported. The crystal structures of four fully protected tripeptides containing the Afc residue in position 2: Z-X(1)-Afc(2)-Y(3)-OMe (peptide a: X = Y = Gly; peptide b: X = Aib, C(alpha, alpha)-dimethylglycine, Y = Gly; peptide c: X = Gly, Y = Aib; peptide d: X = Y = Aib) have been solved by x-ray crystallography. All the results suggest that the Afc residue has a high propensity to assume an extended conformation. In fact, the Afc residue adopts an extended conformation in three peptides examined in this paper (peptides a-c). In contrast, Afc was found in a folded conformation, in the 3(10)-helical region, only in the peptide d, in which it is both preceded and followed by the strong helix promoting Aib.     

Folding intermediates of a model three-helix bundle protein. Pressure and cold denaturation studies

The stability and equilibrium unfolding of a model three-helix bundle protein, alpha(3)-1, by guanidine hydrochloride (GdnHCl), hydrostatic pressure, and temperature have been investigated. The combined use of these denaturing agents allowed detection of two partially folded states of alpha(3)-1, as monitored by circular dichroism, intrinsic fluorescence emission, and fluorescence of the hydrophobic probe bis-ANS (4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfonic acid). The overall free-energy change for complete unfolding of alpha(3)-1, determined from GdnHCl unfolding data, is +4.6 kcal/mol. The native state is stabilized by -1.4 kcal/mol relative to a partially folded pressure-denatured intermediate (I(1)). Cold denaturation at high pressure gives rise to a second partially (un)folded conformation (I(2)), suggesting a significant contribution of hydrophobic interactions to the stability of alpha(3)-1. The free energy of stabilization of the native-like state relative to I(2) is evaluated to be -2.5 kcal/mol. Bis-ANS binding to the pressure- and cold-denatured states indicates the existence of significant residual hydrophobic structure in the partially (un)folded states of alpha(3)-1. The demonstration of folding intermediates of alpha(3)-1 lends experimental support to a number of recent protein folding simulation studies of other three-helix bundle proteins that predicted the existence of such intermediates. The results are discussed in terms of the significance of de novo designed proteins for protein folding studies.     

Conformational behaviour of Cα,α-diphenylglycine: folded vs. extended structures in DϕG-containing tripeptides

The crystal structures of three fully protected tripeptides containing the Dϕg residue (C^α,α-diphenylglycine) in the central position are reported, namely Z-Gly-Dϕg-Gly-OMe ( a ), Z-Gly-Dϕg-Aib-OMe ( b ) and Z-Aib-Dϕg-Aib-OMe ( c ). The molecular conformations are quite unusual because the Dϕg residue adopts a folded conformation in the 3₁₀-helical region when the following residue adopts a folded conformation of opposite handedness (peptides b and c ). In contrast, the Dϕg residue adopts the more frequently observed fully extended conformation when the following residue adopts a semi-extended conformation (peptide a ). These findings are in agreement with the theoretical calculations on Ac-Dϕg-Aib-NHCH₃ and Ac-Aib-Dϕg-NHCH₃ also reported in this work. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Conformational behavior of C alpha,alpha-diphenyl glycine: extended conformation in tripeptides containing consecutive D phi G residues

Recent studies on the conformational preferences of the Dphig (C(alpha,alpha)-diphenylglycine) residue showed that this C(alpha,alpha)-disubstituted glycine has a structural versatility. In fact, depending on the nature of the following or preceding residue, Dphig can assume either folded or extended conformations. We have carried out the analysis of the conformational preferences of the Dphig residue in tripeptides containing consecutive Dphig residues. The crystal structures of Z-Dphig-Dphig -Dphig-OMe (a; Z = benzyloxycarbonyl; OMe = methyl ester), Z-Aib-Dphig-Dphig-OMe (b; Aib = alpha-aminoisobutyric acid), and Z-Ac(3)c-Dphig-Dphig-OMe (c; Ac(3)c = alpha-amino-cyclopropan carboxylic acid), are here reported. The Dphig residues adopt the fully extended conformation in the three tripeptides examined. Together with our previous findings on Dphig containing peptides, the structures of the peptides here examined, indicate that the presence of adjacent Dphig residue in the sequence further stabilizes the extended conformation.     

Phosphorylation of mRNA Decapping Protein Dcp1a by the ERK Signaling Pathway during Early Differentiation of 3T3-L1 Preadipocytes

Background

Turnover of mRNA is a critical step in the regulation of gene expression, and an important step in mRNA decay is removal of the 5′ cap. We previously demonstrated that the expression of some immediate early gene mRNAs is controlled by RNA stability during early differentiation of 3T3-L1 preadipocytes.

Methodology/Principal Findings

Here we show that the mouse decapping protein Dcp1a is phosphorylated via the ERK signaling pathway during early differentiation of preadipocytes. Mass spectrometry analysis and site-directed mutagenesis combined with a kinase assay identified ERK pathway–mediated dual phosphorylation at Ser 315 and Ser 319 of Dcp1a. To understand the functional effects of Dcp1a phosphorylation, we examined protein-protein interactions between Dcp1a and other decapping components with co-immunoprecipitation. Dcp1a interacted with Ddx6 and Edc3 through its proline-rich C-terminal extension, whereas the conserved EVH1 (enabled vasodilator-stimulated protein homology 1) domain in the N terminus of Dcp1a showed a stronger interaction with Dcp2. Once ERK signaling was activated, the interaction between Dcp1a and Ddx6, Edc3, or Edc4 was not affected by Dcp1a phosphorylation. Phosphorylated Dcp1a did, however, enhanced interaction with Dcp2. Protein complexes immunoprecipitated with the recombinant phosphomimetic Dcp1a(S315D/S319D) mutant contained more Dcp2 than did those immunoprecipitated with the nonphosphorylated Dcp1a(S315A/S319A) mutant. In addition, Dcp1a associated with AU-rich element (ARE)-containing mRNAs such as MAPK phosphatase-1 (MKP-1), whose mRNA stability was analyzed under the overexpression of Dcp1a constructs in the Dcp1a knockdown 3T3-L1 cells.

Conclusions/Significance

Our findings suggest that ERK-phosphorylated Dcp1a enhances its interaction with the decapping enzyme Dcp2 during early differentiation of 3T3-L1 cells.     

The prosequence of human lactase-phlorizin hydrolase modulates the folding of the mature enzyme.

The efficient transport of proteins along the secretory pathway requires that the polypeptide adopts a stably folded conformation to egress the endoplasmic reticulum (ER). The transport-competent precursor of the brush border enzyme LPH, pro-LPH, undergoes an intracellular cleavage process in the trans-Golgi network between Arg(734) and Leu(735) to yield LPH beta(initial). The role of the prodomain comprising the N-terminally located 734 amino acids of pro-LPH, LPH alpha, in the folding events of LPH beta(initial) has been analyzed by the individual expression of both forms in COS-1 cells. Following synthesis at 37 degrees C LPH beta(initial) acquires a misfolded and enzymatically inactive conformation that is degraded by trypsin. A temperature shift to 20 degrees C generates a stable, trypsin-resistant, and enzymatically active LPH beta(initial) indicating that the individual expression of LPH beta(initial) results in a temperature-sensitive conformation. This form interacts at non-permissive temperatures sequentially with the ER chaperones immunoglobulin-binding protein and calnexin resulting in an ER retention. The LPH alpha prodomain resides in the ER when individually expressed. It reveals compact structural features that are stabilized by disulfide bridges. LPH alpha and LPH beta(initial) readily interact with each other upon coexpression, and this interaction appears to trigger the formation of a trypsin-resistant, correctly folded, enzymatically active, and transport-competent LPH beta(initial) polypeptide. These data clearly demonstrate that the proregion of pro-LPH is an intramolecular chaperone that is critically essential in facilitating the folding of the intermediate form LPH beta(initial) in the context of the pro-LPH polypeptide.     

Crystal structure of the E. coli dipeptidyl carboxypeptidase Dcp: further indication of a ligand-dependent hinge movement mechanism

Dcp from Escherichia coli is a 680 residue cytoplasmic peptidase, which shows a strict dipeptidyl carboxypeptidase activity. Although Dcp had been assigned to the angiotensin I-converting enzymes (ACE) due to blockage by typical ACE inhibitors, it is currently grouped into the M3 family of mono zinc peptidases, which also contains the endopeptidases neurolysin and thimet oligopeptidase (TOP). We have cloned, expressed, purified, and crystallized Dcp in the presence of an octapeptide "inhibitor", and have determined its 2.0A crystal structure using MAD methods. The analysis revealed that Dcp consists of two half shell-like subdomains, which enclose an almost closed two-chamber cavity. In this cavity, two dipeptide products presumably generated by Dcp cleavage of the octapeptide bind to the thermolysin-like active site fixed to side-chains, which are provided by both subdomains. In particular, an Arg side-chain backed by a Glu residue, together with two Tyr phenolic groups provide a charged anchor for fixing the C-terminal carboxylate group of the P2' residue of a bound substrate, explaining the strict dipeptidyl carboxypeptidase specificity of Dcp. Tetrapeptidic substrates are fixed only via their main-chain functions from P2 to P2', suggesting a broad residue specificity for Dcp. Both subdomains exhibit very similar chain folds as the equivalent but abducted subdomains of neurolysin and TOP. Therefore, this "product-bound" Dcp structure seems to represent the inhibitor/substrate-bound "closed" form of the M3 peptidases, generated from the free "open" substrate-accessible form by a hinge-bending mechanism. A similar mechanism has recently been demonstrated experimentally for ACE2.     

UV resonance Raman investigation of a 3(10)-helical peptide reveals a rough energy landscape

We used UVRRS at 194 and 204 nm excitation to examine the backbone conformation of a 13-residue polypeptide (gp41(659-671)) that has been shown by NMR to predominantly fold into a 3(10)-helix. Examination of the conformation sensitive AmIII(3) region indicates the peptide has significant populations of beta-turn, PPII, 3(10)-helix, and pi-helix-like conformations but little alpha-helix. We estimate that at 1 degree C on average six of the 12 peptide bonds are in folded conformations (predominantly 3(10)- and pi-helix), while the other six are in unfolded (beta-turn/PPII) conformations. The folded and unfolded populations do not change significantly as the temperature is increased from 1 to 60 degrees C, suggesting a unique energy landscape where the folded and unfolded conformations are essentially degenerate in energy and exhibit identical temperature dependences.     

An unusual C-H...O hydrogen bond mediated reversal of polypeptide chain direction in a synthetic peptide helix

An unusual C-terminal conformation has been detected in a synthetic decapeptide designed to analyze the stereochemistry of helix termination in polypeptides. The crystal structure of the decapeptide Boc-Leu-Aib-Val-Ala-Leu-Aib-Val-(D)Ala-(D)Leu-Aib-OMe reveals a helical segment spanning residues 1-7 and helix termination by formation of a Schellman motif, generated by (D)Ala(8) adopting the left-handed helical (alpha(L)) conformation. The extended conformation at (D)Leu(9) results in a compact folded structure, stabilized by a potentially strong C-H. O hydrogen bond between Ala(4) C(alpha)H and (D)Leu(9) CO. The parameters for C-H. O interaction are Ala(4) C(alpha)H. O=C (D)Leu(9) distance 3.27 A, C(alpha)-H. O angle 176 degrees, and O. H(alpha) distance 2.29 A. This structure suggests that insertion of contiguous D-residues may provide a handle for the generation of designed structures containing more than one helical segment folded in a compact manner.     

alpha Domain deletion converts streptokinase into a fibrin-dependent plasminogen activator through mechanisms akin to staphylokinase and tissue plasminogen activator

The mechanism of action of plasminogen (Pg) activators may affect their therapeutic properties in humans. Streptokinase (SK) is a robust Pg activator in physiologic fluids in the absence of fibrin. Deletion of a "catalytic switch" (SK residues 1-59), alters the conformation of the SK alpha domain and converts SKDelta59 into a fibrin-dependent Pg activator through unknown mechanisms. We show that the SK alpha domain binds avidly to the Pg kringle domains that maintain Glu-Pg in a tightly folded conformation. By virtue of deletion of SK residues 1-59, SKDelta59 loses the ability to unfold Glu-Pg during complex formation and becomes incapable of nonproteolytic active site formation. In this manner, SKDelta59 behaves more like staphylokinase than like SK; it requires plasmin to form a functional activator complex, and in this complex SKDelta59 does not protect plasmin from inhibition by alpha(2)-antiplasmin. At the same time, SKDelta59 is unlike staphylokinase or SK and is more like tissue Pg activator, because it is a poor activator of the tightly folded form of Glu-Pg in physiologic solutions. SKDelta59 can only activate Glu-Pg when it was unfolded by fibrin interactions or by Cl(-)-deficient buffers. Taken together, these studies indicate that an intact alpha domain confers on SK the ability to nonproteolytically activate Glu-Pg, to unfold and process Glu-Pg substrate in physiologic solutions, and to alter the substrate-inhibitor interactions of plasmin in the activator complex. The loss of an intact alpha domain makes SKDelta59 activate Pg through classical "fibrin-dependent mechanisms" (akin to both staphylokinase and tissue Pg activator) that include: 1) a marked preference for a fibrin-bound or unfolded Glu-Pg substrate, 2) a requirement for plasmin in the activator complex, and 3) the creation of an activator complex with plasmin that is readily inhibited by alpha(2)-antiplasmin.     

Dcp2 Decaps m2,2,7GpppN-capped RNAs, and its activity is sequence and context dependent

Hydrolysis of the mRNA cap plays a pivotal role in initiating and completing mRNA turnover. In nematodes, mRNA metabolism and cap-interacting proteins must deal with two populations of mRNAs, spliced leader trans-spliced mRNAs with a trimethylguanosine cap and non-trans-spliced mRNAs with a monomethylguanosine cap. We describe here the characterization of nematode Dcp1 and Dcp2 proteins. Dcp1 was inactive in vitro on both free cap and capped RNA and did not significantly enhance Dcp2 activity. Nematode Dcp2 is an RNA-decapping protein that does not bind cap and is not inhibited by cap analogs but is effectively inhibited by competing RNA irrespective of RNA sequence and cap. Nematode Dcp2 activity is influenced by both 5' end sequence and its context. The trans-spliced leader sequence on mRNAs reduces Dcp2 activity approximately 10-fold, suggesting that 5'-to-3' turnover of trans-spliced RNAs may be regulated. Nematode Dcp2 decaps both m(7)GpppG- and m(2,2,7)GpppG-capped RNAs. Surprisingly, both budding yeast and human Dcp2 are also active on m(2,2,7)GpppG-capped RNAs. Overall, the data suggest that Dcp2 activity can be influenced by both sequence and context and that Dcp2 may contribute to gene regulation in multiple RNA pathways, including monomethyl- and trimethylguanosine-capped RNAs.     

The complete chirospectroscopic signature of the peptide 3(10)-helix in aqueous solution

We synthesized by solution methods a water-soluble, terminally blocked heptapeptide based on five markedly helicogenic, C(alpha)-tetrasubstituted alpha-amino acids C(alpha)-methyl-L-norvalines and two strongly hydrophilic 2-amino-3-[1-(1,4,7-triazacyclononane)]-L-propanoic acid residues at positions 2 and 5. A Fourier transform infrared absorption and NMR analysis in deuterated chloroform and aqueous solutions of the heptapeptide and two side-chain protected synthetic precursors confirmed our working hypothesis that all oligomers are folded in the 3(10)-helical conformation. Based on these findings, we exploited this heptapeptide as a chiral reference compound for detailed electronic CD, vibrational CD, and Raman optical activity characterizations of the 3(10)-helix in aqueous solution.     

Structure of the N-terminal extension of human aspartyl-tRNA synthetase: implications for its biological function

Human aspartyl-tRNA synthetase (hDRS) contains an extension at the N-terminus, which is involved in the transfer of Asp-tRNA to elongation factor alpha1 (EF1alpha). The structure of the N-terminal extension is critical to its function. Conformational studies on the synthetic, 21-residue N-terminal extension peptide (Thr5-Lys25) of human aspartyl-tRNA synthetase using 1H nuclear magnetic resonance (NMR) spectroscopy, showed that the C-terminus adopts a regular alpha-helix with amphiphilicity, while the N-terminus shows a less-ordered structure with a flexible beta-turn. The observed characteristics suggest a structural switch model, such that when the tRNA is in the stretched conformation, the peptide reduces the rate of dissociation of Asp-tRNA from human aspartyl-tRNA synthetase, and provides enough time for elongation factor 1alpha to interact with the Asp-tRNA. Following Asp-tRNA transfer to EF1alpha, the peptide assumes the folded conformation. The structural switch model supports the direct transfer mechanism.     

Conformations of dehydrophenylalanine containing peptides: nmr studies of an acyclic hexapeptide with two delta Z-Phe residues

The conformation of an acyclic dehydrophenylalanine (delta Z-Phe) containing hexapeptide, Boc-Phe-delta Z-Phe-Val-Phe-delta Z-Phe-Val-OMe, has been investigated in CDCl3 and (CD3)2SO by 270-MHz 1H-nmr. Studies of NH group solvent accessibility and observation of interresidue nuclear Overhauser effects (NOEs) suggest a significant solvent-dependent conformational variability. In CDCl3, a population of folded helical conformations is supported by the inaccessibility to solvent of the NH groups of residues 3-6 and the detection of several NiH----Ni + 1H NOEs. Evidence is also obtained for conformational heterogeneity from the detection of some Ci alpha H----Ni + 1H NOEs characteristic of extended strands. In (CD3)2SO, the peptide largely favors an extended conformation, characterized by five solvent-exposed NH groups and successive Ci alpha H----Ni + 1H NOEs for the L-residues and Ci beta H----Ni + 1H NOEs for the delta Z-Phe residues. The results suggest that delta Z-Phe residues do not provide compelling conformational constraints.     

Role of peptide backbone conformation on biological activity of chemotactic peptides

To investigate the role of peptide backbone conformation on the biological activity of chemotactic peptides, we synthesized a unique analog of N-formyl-Met-Leu-Phe-OH incorporating the C alpha,alpha disubstituted residue, dipropylglycine (Dpg) in place of Leu. The conformation of the stereochemically constrained Dpg analog was examined in the crystalline state by x-ray diffraction and in solution using NMR, IR, and CD methods. The secretagogue activity of the peptide on human neutrophils was determined and compared with that of a stereochemically constrained, folded type II beta-turn analog incorporating 1-aminocyclohexanecarboxylic acid (Ac6c) at position 2 (f-Met-Ac6c-Phe-OMe), the parent peptide (f-Met-Leu-Phe-OH) and its methyl ester derivative (f-Met-Leu-Phe-OMe). In the solid state, the Dpg analog adopts an extended beta-sheet-like structure with an intramolecular hydrogen bond between the NH and CO groups of the Dpg residue, thereby forming a fully extended (C5) conformation at position 2. The phi and psi values for Met and Phe residues are significantly lower than the values expected for an ideal antiparallel beta conformation causing a twist in the extended backbone both at the N and C termini. Nuclear magnetic resonance studies suggest the presence of a significant population of the peptide molecules in an extended antiparallel beta conformation and the involvement of Dpg NH in a C5 intramolecular hydrogen bond in solutions of deuterated chloroform and deuterated dimethyl sulfoxide. IR studies provide evidence for the presence of an intramolecular hydrogen bond in the molecule and the antiparallel extended conformation in chloroform solution. CD spectra in methanol, trifluoroethanol, and trimethyl phosphate indicate that the Dpg peptide shows slight conformational flexibility, whereas the folded Ac6c analog is quite rigid. The extended Dpg peptide consistently shows the highest activity in human peripheral blood neutrophils, being approximately 8 and 16 times more active than the parent peptide and the folded Ac6c analog, respectively. However, the finding that all four peptides have ED50 (the molar concentration of peptide to induce half-maximal enzyme release) values in the 10(-8)-10(-9) M range suggests that an induced fit mechanism may indeed be important in this ligand-receptor interaction. Moreover, it is also possible that alterations in the backbone conformation at the tripeptide level may not significantly alter the side chain topography and/or the accessibility of key functional groups important for interaction with the receptor.