NMR Structure of the First Extracellular Domain of Corticotropin-releasing Factor Receptor 1 (ECD1-CRF-R1) Complexed with a High Affinity Agonist^*

Protein Expression and Purification

The purification and labeling of the ECD1-CRF-R1 expressed in Escherichia coli were carried out as described previously (15, 29).

Synthesis of αhcCRF, with C-13 and N-15 Isotopically Labeled Amino Acids

The synthesis was performed on a methyl-benzhydrylamine resin using the Fmoc (N-(9-fluorenyl)methoxycarbonyl) strategy. Purification and characterization used established procedures (see Refs. 30, 31). Full details are given in the Supplemental Information section.

Radioreceptor Assays

Radioreceptor assays for full-length receptors expressed in mammalian cells were carried out as described previously (14) and for soluble receptors as described previously (32).

NMR Experiments and Analysis

All NMR spectra were recorded at 35 °C using a Bruker 700-MHz spectrometer equipped with four radiofrequency channels and a triple resonance cryo-probe with shielded z-gradient coil. The NMR samples contained either 0.3 mm ¹³C,¹⁵N-labeled ECD1-CRF-R1 and an equimolar concentration of unlabeled αhcCRF or 0.3 mm ¹³C,¹⁵N-labeled αhcCRF (labeled uniformly by ¹⁵N and ¹³C except for residues Thr¹¹, Glu³⁰, and Lys³³) and an equimolar concentration of unlabeled ECD1-CRF-R1 in 10 mm BisTris(HCl), 95% H₂O, 5% D₂O, pH 6.5. The sample for the two-dimensional ¹H,¹H NOESY was prepared with an equimolar concentration of 0.4 mm of unlabeled ECD1-CRF-R1 and αhcCRF, respectively, in 10 mm BisTris(HCl), pH 6.5, in 100% D₂O.

Sequential assignment was performed with the standard protocol for ¹³C,¹⁵N-labeled samples (33). ¹H,¹³C, and ¹⁵N backbone resonances were assigned using triple resonance HNCA (34), HNCACB (35), and three-dimensional ¹⁵N-resolved ¹H,¹H NOESY experiments (36). The side-chain ¹H resonances were assigned using HCCH correlation spectroscopy (37) and ¹³C-resolved ¹H,¹H NOESY (38) experiments. Aromatic side-chain assignments were obtained with two-dimensional ¹³C,¹H HSQC, two-dimensional ¹H,¹H NOESY (39) in D₂O, and three-dimensional ¹³C aromatic-resolved ¹H,¹H NOESY (38) experiments. Distance constraints for the structure calculation were derived from three-dimensional ¹³C,¹⁵N-resolved ¹H,¹H NOESY and two-dimensional ¹H,¹H standard NOESY (39) spectra recorded with mixing times of either 100 or 120 ms. For all of the spectra quadrature detection in the indirect dimensions was achieved using States-TPPI (40). The water signal was suppressed using spin-lock pulses (41) or the WATERGATE sequence (42). All of the spectra were processed with the program PROSA (43) and were analyzed with the program CARA (44).

Determination of the Structure of ECD1-CRF-R1 Complexed with αhcCRF

Meaningful distance restraints (∼1795) and angle restraints (371) were collected for the calculation of the structure of the complex (Table 1). These structural restraints were used as an input for the structure calculation with the program CYANA (43) followed by restrained energy minimization using CNS (45). A total of 100 conformers was initially generated by CYANA, and the bundle of 20 conformers with the lowest target function was used to represent the three-dimensional NMR structure. The structures are validated with the program PROCHECK (46), and the structure has been deposited in the Protein Data Bank data base with code 2L27.

Selection of a Soluble High Affinity Agonist

A major challenge in the three-dimensional structure determination of ECD1-CRF-R1 complexed with an agonist was to obtain a high affinity agonist that was soluble above pH 5 at concentrations required for the NMR experiments. Comparison of several CRF analogs showed that the novel CRF agonist, αhcCRF, satisfied the requirements, i.e. it bound with low nanomolar affinity to both CRF-R1 (∼1 nm) and the ECD1-CRF-R1 (∼30 nm, Table 2) and was soluble at physiological pH. This peptide is in part only related to α-helical-CRF(1–41) and to astressin (3, 47). The sequence of αhcCRF is cyclo(30–33)Ac-PP⁵ISLDL¹⁰TFNLL¹⁵REVLE²⁰IAKAE²⁵QEAEE³⁰AAK³³NR³⁵LLLEE⁴⁰A-NH₂. The lactam bridge connecting the side chains of residues Glu³⁰ and Lys³³ of αhcCRF stabilizes the α-helical domain first established for the antagonist astressin (3).

Slow Conformational Exchange Dynamics of Free ECD1-CRF-R1

The NMR structure of the free ECD1-CRF-R1 was not determined because resonances for many residues were not observed in the ¹⁵N,¹H TROSY spectrum (Fig. 2A). These include the N-terminal residues 1–24 and the C-terminal residues 110–127 of the construct. In addition, several residues that are part of loop 2 involved in ligand binding (see below) (i.e. Arg⁶⁶, Cys⁶⁸, Phe⁷¹, Phe⁷², Gly⁷⁴, Val⁷⁵, Tyr⁷⁷, Asn⁷⁸, and Ala⁹⁵) were absent in the ¹⁵N,¹H TROSY spectrum (Fig. 2A). Changes in pH or temperature failed to improve the quality of the spectrum. The absence of these peaks in the spectrum may be due to slow conformational exchange dynamics involving the backbone, which results in line broadening so severe that peaks cannot be detected. A similar observation was documented for the free ECD1-CRF-R2β, but it was limited to only a few residues located in loop 2 (i.e. Tyr⁸⁷ (corresponding to Phe⁷¹ in CRF-R1), Phe⁸⁸ (Phe⁷²); Asn⁸⁹ (Tyr⁷³), Gly⁹⁰ (Gly⁷⁴), Ile⁹¹ (Val⁷⁵), Lys⁹² (Arg⁷⁶), Arg⁹⁷ (Asn⁸¹)) (2). For the ECD1-CRF-R2β, this slow conformational exchange was estimated to be on the time scale of 10⁻² s, and a similar rate was also estimated for the ECD1-CRF-R1. However, because the number of cross-peaks absent in the ¹⁵N,¹H TROSY spectrum is much larger for ECD1-CRF-R1 than for ECD1-CRF-R2β, the conformational exchange dynamics of ECD1-CRF-R1 must be of greater amplitude and/or must involve a larger segment of the ECD1-CRF-R1 compared with ECD1-CRF-R2β. For ECD1-CRF-R2β, this slow conformational exchange phenomenon was suppressed, at least in part, when complexed with the antagonist astressin, because all of the missing cross-peaks of the amide moieties appeared in the ¹⁵N,¹H TROSY spectrum of the complex. Assuming that all of the peaks might appear in the spectrum of ECD1-CRF-R1 complexed with the agonist, we carried out the structural studies of the ECD1-CRF-R1 complexed with αhcCRF.

Mapping the Agonist-binding Site on ECD1-CRF-R1 by Agonist-induced Chemical Shift Changes

Insight into the agonist-binding site on ECD1-CRF-R1 can be obtained from an analysis of the resonance shifts in the NMR spectra upon addition of αhcCRF (34). Fig. 2A shows the ¹⁵N,¹H TROSY spectrum of the ECD1-CRF-R1 in the absence and presence of equimolar αhcCRF. Following agonist binding, large chemical shift changes of resonances or appearance of cross-peaks was observed at four different regions of the ECD1 as follows: (i) Leu⁵⁰ and Ile⁵¹, Val⁶⁵; (ii) Arg⁶⁶, Cys⁶⁸, Phe⁷¹–Asn⁷⁸; (iii) Asn⁸², Gly⁸³, Arg⁸⁵, Ala⁹⁵–Ser¹⁰⁰; and (iv) Gln¹⁰³–Glu¹⁰⁸ (Fig. 2B). Most of these residues are of hydrophobic nature and conserved between the two CRF receptors (Ile⁵¹, Arg⁶⁶, Cys⁶⁸, Phe⁷², Gly⁷⁴, Val⁷⁵, Tyr⁷⁷, Asn⁷⁸, Asn⁹⁸, Tyr⁹⁹) (Fig. 2C). This structural study, together with similar chemical shift perturbation data observed for the ECD1-CRF-R2β-astressin complex (2), suggests that both the agonist and antagonist interact with the same residues in the ECD1s.

Three-dimensional Structure of the ECD1-CRF-R1 Complexed with αhcCRF

Almost complete sequential assignment of the various resonances of the ECD1-CRF-R1-αhcCRF complex was obtained using standard procedures, and the three-dimensional structure was determined (see under “Materials and Methods”). The good quality of the three-dimensional structure is represented by the small root mean square deviation of 0.88 Å for residues 42–105 of the ECD1 and for residues 27–38 of αhcCRF (Fig. 3, A and B), as well as by the small value of residual constraint violations in the 20 refined conformers, and by the small deviations from ideal geometry (Table 1). In addition, the input data represent a self-consistent set, and the restraints are well satisfied in the calculated conformers, and similar energy values were obtained for all of the 20 conformers.

As documented already in the structures of the ECD1 of CRF-R2β (2, 29) and of the ECD1s of the other members of the B1 receptor family (25–28), the overall fold of the ECD1 of CRF-R1 is the short consensus repeat (SCR) motif with the three disulfide bonds between cysteine residues, Cys³⁰–Cys⁵⁴, Cys⁴⁴–Cys⁸⁷, and Cys⁶⁸–Cys¹⁰². The SCR includes a short N-terminal α-helix (Asp²⁷–Glu³¹) and two anti-parallel β-sheet regions around residues Ser⁴⁷–Val⁴⁸ (β1 strand), Cys⁵⁴–Trp⁵⁵ (β2 strand), Leu⁶³–Arg⁶⁶ (β3 strand), and Gly⁸³–Glu⁸⁶ (β4 strand). The chemical shift values of ¹³C^α, ¹³C^β, and ¹H^α support the presence of these secondary structural elements. At the core of the SCR motif, the aliphatic side chain of the conserved Arg⁸⁵ is sandwiched between the highly conserved Trp⁵⁵ and Trp⁹³ residues and is in close proximity to the side chain of the conserved Asp⁴⁹ and thus may possibly be involved in a salt bridge interaction (Fig. 3B). Furthermore, the indole nitrogen of Trp⁵⁵ forms a hydrogen bond with the carboxyl group of Asp⁴⁹ stabilizing the β1-β2 hairpin (Fig. 3B). These structural features are very similar to those observed for the ECD1 of CRF-R2β and are supported by the up-field shifted side chain resonances of Arg⁸⁵ (βCH₂, 0.42 and −0.55; γCH₂, 0.65 and 1.36; δCH₂, 0.97 and 0.18 ppm) that require close proximity to an aromatic side chain because aromatic ring currents cause such high field shifts. Additional up-field shifted resonances are observed for the methyl protons of Ile⁵¹ (0.12 and −0.28 ppm), Thr⁵³ (0.08 ppm), the α-protons of Gly⁸³ (4.42 and 2.92 ppm), and methyl protons of Val⁹⁷ (0.41 and −0.29 ppm), and they are all attributed to the close proximity of the residues to the aromatic ring of Tyr⁹⁹.

Conformation of the Agonist αhcCRF Both Free and Complexed with the ECD1-CRF-R1

For the free ¹⁵N,¹³C-labeled αhcCRF (amino acid numberings per Fig. 4G) at pH 6.5 and 298 K, all of the expected 30 peaks were observed in the ¹⁵N,¹H HMQC spectrum (Thr¹¹, Glu³⁰, and Lys³³ were not labeled, see under “Materials and Methods”) (Fig. 4A, red contours). In addition to the major conformation, a minor conformation (∼⅓ of the major conformation) is also present for most of the N-terminal residues up to residue Leu¹⁰. This minor conformation is attributed to cis/trans isomerizations of the N-terminal proline residues. The chemical shift values for the C^α, C^β, and H^α protons (Fig. 4, C and D, red bars) of the free peptide indicate that the peptide segment from residues Phe¹² to Leu³⁷ is partially in a helical conformation.

Fig. 4A also shows the ¹⁵N,¹H HMQC spectrum of ¹³C,¹⁵N-labeled αhcCRF complexed with unlabeled ECD1-CRF-R1 (black contours). When compared with the ¹⁵N,¹H HMQC spectrum of free αhcCRF, two prominent features in the complex spectrum are observed as follows: (i) the larger dispersion of the cross-peaks attributed to a higher ordered structure of αhcCRF in the complex, and (ii) broad cross-peaks with large line widths due to the larger size of the complex and/or due to slow conformational exchange. Most prominent chemical shift changes as well as line broadening are observed for the C-terminal residues of αhcCRF with the maximum shift for Ala⁴¹ (Fig. 4E). The chemical shifts for the C^α, C^β, and H^α protons (Fig. 4, C and D, black bars) suggest that αhcCRF prefers a helical conformation when bound to the ECD1-CRF-R1 and that the helicity is more pronounced upon complex formation not only at the C terminus but also toward the N terminus of the peptide (Fig. 4, A and B). This is further supported by the α-helical NOEs (i.e. αβ(i,i + 3), αN(i,i + 3), and αN(i,i + 4)) observed for residues Phe¹² to Ala⁴¹ (Fig. 4G). In addition, amide proton chemical shifts between Ala³¹ and Ala⁴¹ (Fig. 4F) show a wave-like pattern attributed to the amphipathic nature of the helix in the complex, observed also for astressin bound to the ECD1 of CRF-R2β (2). In addition, a kink of the long helix is observed between residues Glu²⁷ to Glu²⁹ of αhcCRF (Fig. 3). The positioning of this kink is similar to the kinks observed for CRF family ligands in the solvent DMSO (48) but is absent in all the other ligands of family B1 when they are bound to their respective ECD1s in the crystal structures. Although the role of this kink may be understood only in the context of the full-length receptor, it enlarges the conformational space of the N-terminal peptide segment, which is possibly important for receptor-specific signaling (Fig. 3A) (49, 50).

Molecular Interactions between αhcCRF and the ECD1

The helical segment of αhcCRF bound to the ECD1 is along the hydrophobic face of the protein, covering an area of 2647 Ų of the ECD1 (Fig. 5). The C-terminal αhcCRF residues Leu³⁷, Leu³⁸, and Ala⁴¹ are involved in hydrophobic contacts with Phe⁷², Tyr⁷³, Tyr⁷⁷, Ile⁵¹, Cys⁶⁸, Pro⁶⁹, Val⁹⁷, Cys¹⁰², and Tyr⁹⁹ of the ECD1. In particular, the side chain of Leu³⁸ is located in a deep hydrophobic pocket surrounded by Ile⁵¹, Cys⁶⁸, Pro⁶⁹, Phe⁷², Tyr⁷⁷, Tyr⁹⁹, and Cys¹⁰² of the ECD1 (note that up-field shifted resonances for the β and methyl protons of Leu³⁸ are indicative of its interactions with aromatic side chains of the ECD1). The amide group at the C terminus of αhcCRF is involved in an inter-molecular hydrogen bond with the backbone carbonyl of Val⁹⁷. In return, the backbone amide proton of Val⁹⁷ is involved in a hydrogen bond with the backbone carbonyl of Ala⁴¹ in αhcCRF (Fig. 5). These hydrogen bonds explain the necessity of C-terminal amidation for high affinity recognition of CRF ligands (48, 51). Although there are several NOEs observed between the side chain of Asn³⁴ to Tyr⁷³ and Val⁷⁵, in the three-dimensional structure, these two side chains are not close enough to form an intermolecular hydrogen bond. The side chain of Ala³¹ of αhcCRF also interacts with Val⁷⁵ and Tyr⁷⁷ through hydrophobic interactions. The side chain of Arg³⁵ is in close proximity to the side chain of Glu¹⁰⁴, thereby facilitating a salt bridge interaction. Because the side chain resonances of Arg³⁵ could not be observed in the ¹³C-resolved ¹H,¹H NOESY spectrum except for the δ protons, its interaction must be interpreted with care.

Comparison of the Structures of ECD1-CRF-R1 Complexed with the Agonist αhcCRF and ECD1-CRF-R2β Complexed with the Antagonist Astressin

Recently, our group reported the structure of the ECD1 of CRF-R2β complexed with the peptide antagonist astressin (2). Comparison of the structures of the complexes of ECD1-CRF-R1 and ECD1-CRF-R2β enables the identification of the common interaction sites. Both of the structures have the SCR motif characteristic of the ECD1s of family B1 members (Fig. 6A). There is a short N-terminal helix observed for ECD1-CRF1-R1, which was absent in the structure of ECD1-CRF-R2β because in the latter the N-terminal segment was truncated in the protein construct. Although conformation of loop 1 is not defined in either ECD1, loops 2 and 3 are structured and interact with the corresponding ligand in a slightly different manner (Fig. 6B). The backbone of loop 2 of ECD1-CRF-R2β folds closer to the ligand than the corresponding loop in ECD1-CRF-R1, although the opposite holds for loop 3.

There are also structural differences of the ligand. Although both the agonist and the antagonist bind to almost the same region of the ECD1, the orientations of the ligands are slightly different with respect to loop 2 (Fig. 6, B–D). Furthermore, the C-terminal residues of astressin prefer a 3₁₀ helix, whereas in αhcCRF, they are in an α-helical conformation. In both ligands, the backbone carbonyl of the last residue (Ala/Ile⁴¹) is involved in a hydrogen bond with the amide proton of Val^97/113. The side chains of the ligand residues, Glu³⁹, Leu/Lys³⁶, Ala/His³², and Gln²⁹ are completely solvent-exposed in both of the structures. Also, the side chain of Glu⁴⁰ of αhcCRF is completely solvent-exposed, whereas Ile⁴⁰ of astressin interacts with Gln⁶⁶ of CRF-R2β. The side chain of Leu/Nle³⁸ is completely buried in a hydrophobic core in both structures (Fig. 6). Although the side chains of Phe^72/88 of ECD1 are conserved in their positions and these residues interact with residues Leu³⁷ and Asn³⁴ of the ligand in both of the structures, the positions of Tyr⁷³/Asn⁸⁹ and Tyr^77/93 are distinct. The orientation of Tyr⁷⁷ of CRF-R1 is toward the ligand, whereas in CRF-R2β, Tyr⁹³ points away from the ligand. Consistent with this is the observation that the mutation Y77A in CRF-R1 abrogated high affinity binding of both astressin and sauvagine and significantly increased the EC₅₀ for sauvagine- and urocortin1-stimulated intracellular cAMP accumulation (data not shown). The side chain of Asn³⁴ of astressin is in close proximity to the ECD1 to form a hydrogen bond with the backbone carbonyl of Phe⁸⁸ in ECD1-CRF-R2β. Such a hydrogen bond is missing in the structure of the ECD1-CRF-R1 in complex with αhcCRF. The residues Val⁷⁵/Ile⁹¹ are in almost the same position, and they interact with Ala³¹ of the ligand. The side chain of Arg³⁵ does not directly interact with Glu¹⁰⁴ of the ECD1-CRF-R1, although it is close enough to form a solvent-exposed intermolecular salt bridge. In contrast, in ECD1-CRF-R2β, Arg³⁵ is involved in a buried salt bridge with Glu⁸⁶, which, in ECD1-CRF-R1, is replaced by Ala⁷⁰, and its side chain is solvent-exposed.

Comparison of the NMR Structure of ECD1-CRF-R1-αhcCRF Complex and X-ray Crystallographic Structures of ECD1-CRF-R1 Peptide Complex

The NMR structure of ECD1-CRF-R1 complexed with the high affinity agonist αhcCRF may be compared with the crystal structures of ECD1-CRF-R1 complexed with the low affinity fragments CRF(22–41) and CRF(27–41), which are presumed to be antagonists (1). Interestingly, the ECD1 of CRF-R1 was crystallized in a ligand-dependent manner in three different forms that differ mainly in the conformations of loop 2, to which the ligand binds (Fig. 7). Pioszak et al. (1) suggest that crystal form II is possibly more relevant, physiologically, than the others because only in crystal form II is loop 2 unhindered from crystal packing constraints. Indeed, our NMR structure of ECD-CRF-R1-αhcCRF (Fig. 7, E and F) is more similar to the crystal form II than to forms I and III (Fig. 7, A–D) (1). The differences between the NMR and x-ray structures are discussed in detail in the supplemental material. The comparison between all of the four structures suggests the presence of a structural plasticity of the SCR motif of the ECD1-CRF-R1 (see below).

Structural Plasticity of Both the ECD1 and the Ligand

The absence of cross-peaks for amide moieties of residues in loop 2 in the ¹⁵N,¹H TROSY spectra of both of the ECD1s of CRF-R1 and CRF-R2β suggests the presence of slow conformational exchange dynamics in the ECD1s of CRF receptors. Although the cross-peaks appear in the ¹⁵N,¹H TROSY spectra in the complex with the peptide for both receptor studies, the significant line broadening observed for these peaks suggests that the conformational exchange is only partially suppressed. The amide moieties of the ligand αhcCRF also show broad, weak cross-peaks in the ¹⁵N,¹H HMQC spectra upon complex formation (Fig. 4). The presence of slow conformational exchange dynamics in the millisecond time range for segments of both the ECD1 and the ligand is in agreement with the presence of conformational heterogeneity observed in both the NMR and x-ray structures. Furthermore, the nanomolar binding affinity of the antagonist astressin for a CRF-R2β mutant whose corresponding ECD1 shows molten globule-like conformational states (52) supports the dynamic character of the ECD1. Although we can only speculate about the biological role of these conformational exchange dynamics, their presence could account for the multiple recognition, binding, and signaling observed for the various hormone ligands.

Refinement of the Two-step Binding Mechanism of CRF Peptides to Its Receptors

The two-step model for ligand binding and signaling of type B1 GPCRs (29, 53, 54) proposes that the C-terminal segment of the ligand binds to the ECD1, which then may position the N-terminal portion of the peptide hormone in close proximity to the serpentine regions of the receptor to initiate signaling. The ECD1 is therefore the major peptide-binding domain, and conversely, the C-terminal segment of the ligand is important for high binding affinity and selectivity to the receptors. All of the three-dimensional structures of the ECD1-receptor-ligand complexes are consistent with this model, because the C-terminal segment of the peptide ligand interacts with the ECD1 (1, 2, 26–28). Our NMR studies of the complex between ECD1-CRF-R1 and an agonist further indicate that the recognition of the ligand by the ECD1 not only binds the hormone and positions its N-terminal residues for signal activation but also induces helix formation toward the N terminus of the ligand to generate a conformationally active state. In a recent review (55), this additional function in receptor activation of the ECD1 in family B1 GPCRs was proposed to be based on the following: (i) a structural comparison between various hormone ligands, free and complexed with ECD1s and (ii) the importance of helix-capping residues in the N-terminal region of the numerous corresponding ligands (56–58). The induction of the helix in the ligand upon complex formation with the ECD1 is accompanied by a kink between residues Glu²⁴ to Glu²⁶ of αhcCRF (Figs. 3A and 8); this kink may also play a role in receptor activation by either enlarging the conformational space of the N-terminal peptide segment or by positioning the ECD1 relative to the serpentine region of the receptor important for signaling and/or co-receptor interactions (29). Hence, our three-dimensional structure of the ECD1 of CRF-R1 complexed with an agonist suggests a refined two-step model for receptor activation.

Agonist Versus Antagonist

Previous studies on chemically modified and truncated CRF ligands showed that the first seven residues at the N termini of CRF are not necessary for GPCR signaling (3, 4) and that residue 8 was critical, whereas the C-terminal (∼15) residues are important for binding (47, 53, 54). Hence, CRF analogs truncated by 8 residues or more at the N terminus are antagonists. This finding can easily be understood with the help of the two-step model for ligand binding and signaling of type B1 GPCR discussed above (16, 19, 29). If the N-terminal segment of the ligand is missing, the C-terminal fragment still binds to the receptor but is not able to produce activation. Such a ligand is then evidently an antagonist because it occupies the major binding site thereby blocking peptide agonist binding. The three-dimensional structure of the ECD1-CRF-R1 complexed with the agonist αhcCRF presented here is the first direct experimental proof that supports this hypothesis (Figs. 7 and 8) and shows clearly the similarity of the C-terminal binding of the peptide agonists and antagonists.