Solution structure and mutational analysis of pituitaryadenylate cyclase-activating polypeptide bindingto the extracellular domain of PAC1-RSChaohong Sun, Danying Song, Rachel A. Davis-Taber, Leo W. Barrett, Victoria E. Scott, Paul L. Richardson,Ana Pereda-Lopez, Marie E. Uchic, Larry R. Solomon, Marc R. Lake, Karl A. Walter, Philip J. Hajduk,and Edward T. Olejniczak*

Global Pharmaceutical Discovery Division, Abbott Laboratories, Abbott Park, IL 60064

Edited by Adriaan Bax, National Institutes of Health, Bethesda, MD, and approved March 27, 2007 (received for review December 21, 2006)

The pituitary adenylate cyclase-activating polypeptide (PACAP)receptor is a class II G protein-coupled receptor that contributes tomany different cellular functions including neurotransmission,neuronal survival, and synaptic plasticity. The solution structure ofthe potent antagonist PACAP (residues 6�–38�) complexed to theN-terminal extracellular (EC) domain of the human splice varianthPAC1-R-short (hPAC1-RS) was determined by NMR. The PACAPpeptide adopts a helical conformation when bound to hPAC1-RS

with a bend at residue A18� and makes extensive hydrophobic andelectrostatic interactions along the exposed �-sheet and intercon-necting loops of the N-terminal EC domain. Mutagenesis data onboth the peptide and the receptor delineate the critical interactionsbetween the C terminus of the peptide and the C terminus of theEC domain that define the high affinity and specificity of hormonebinding to hPAC1-RS. These results present a structural basis forhPAC1-RS selectivity for PACAP versus the vasoactive intestinalpeptide and also differentiate PACAP residues involved in bindingto the N-terminal extracellular domain versus other parts of thefull-length hPAC1-RS receptor. The structural, mutational, andbinding data are consistent with a model for peptide binding inwhich the C terminus of the peptide hormone interacts almostexclusively with the N-terminal EC domain, whereas the centralregion makes contacts to both the N-terminal and other extracel-lular parts of the receptor, ultimately positioning the N terminus ofthe peptide to contact the transmembrane region and result inreceptor activation.

NMR � vasoactive intestinal peptide � G protein-coupled receptor

Seven transmembrane domain G protein-coupled receptors(GPCRs) are cell surface proteins that transduce signals

initiated by hormones or neurotransmitters into the cell (1).Class II GPCRs are a family of receptors that bind structurallyrelated peptide hormones including glucagon, glucagon-likepeptides, vasoactive intestinal peptide (VIP), corticotrophin-releasing factor (CRF), parathyroid hormone (PTH), and pitu-itary adenylate cyclase-activating polypeptide (PACAP) hor-mone. This family of receptors is capable of regulatingintracellular concentrations of cAMP through activation of theadenylate cyclase pathway, and some can also modulate intra-cellular calcium levels through the phospholipase C pathway.Structurally, they have low homology with other GPCR familiesbut are well conserved within the family. They all contain arelatively large amino-terminal extracellular (EC) domain thatplays a critical role in ligand binding. The N-terminal EChormone-binding domains have common features, typically con-taining six conserved cysteine residues, two conserved trypto-phan residues, and an aspartate residue which has been sug-gested to be critical for ligand binding (2, 3). Many of the peptideligands of these receptors have related sequences and can bindto more than one receptor subtype (4).

VIP and PACAP are two prototypical neuropeptides thatmodulate Class II GPCRs. They are widely expressed in thecentral and peripheral nervous system and modulate neurotrans-mission, secretagogue, neuroprotective, neurotrophic, and mi-togenic functions (3, 5). Mature VIP exists as a 28-aa peptide,whereas PACAP has two active forms, PACAP-38 and PACAP-27. Mutagenesis studies indicate that deleting the first 5 aa ofPACAP-38 converts PACAP from an agonist of the receptor toan antagonist with similar binding affinity. These observationsare consistent with the model in which the N terminus of thepeptide is involved in receptor signaling, whereas the remainderof the peptide is important for high affinity binding and receptorspecificity (3, 5).

Two subtypes of receptors have been identified for PACAPand VIP (3, 5). The subtype I receptor (e.g., PAC1-R) is specificfor PACAP over VIP, with a �1,000-fold difference in affinity.In contrast, subtype II receptors (e.g., VPAC1-R and VPAC2-R)bind PACAP and VIP with similar affinities. PAC1-R expressionhas been detected in the central and peripheral nervous system,the limbic system, and the adrenal gland (6) and has beenproposed as a potential target for the treatment of epilepsy,neurodegeneration, and cognition disorders (e.g., Alzheimer’sdisease, schizophrenia, and anxiety) (7). VIP and PACAP alsoregulate the expression and release of proinflammatory cyto-kines and chemokines, and have the potential to be targeted forthe treatment of inflammatory diseases such as asthma (8),Crohn’s disease (9), rheumatoid arthritis (10), and multiplesclerosis (11). PACAP is also up-regulated in models of painafter insult or injury, and thus has been proposed as a potentialtarget for pain modulation (12).

The sequence conservation in both endogenous peptide hor-mones and class II GPCRs suggests a similar mechanisms forsignal transduction. However, the interactions between thepeptides and the receptors are complex and not well understood.Some structural data exist to aid in understanding the complex

Author contributions: C.S., D.S., R.A.D.-T., L.W.B., V.E.S., P.L.R., A.P.-L., M.E.U., L.R.S., M.R.L.,K.A.W., and E.T.O. performed research; and C.S., R.A.D.-T., V.E.S., P.J.H., and E.T.O. wrotethe paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactiveintestinal peptide; GPCR, G protein-coupled receptor; EC, extracellular; NMR, nuclearmagnetic resonance; NOE, Nuclear Overhauser effect; TRNOE, transferred NOE; RDC,residual dipolar coupling.

Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,www.pdb.org (PDB ID code 2JOD) and the BioMagResBank database, www.bmrb.wisc.edu(BMRB ID code 15166).

*To whom correspondence should be addressed at: Abbott Laboratories, 100 Abbott ParkRoad, R46Y, AP10, Abbott Park, IL 60064-6098., E-mail: edward.olejniczak@abbott.com.

This article contains supporting information online at www.pnas.org/cgi/content/full/0611397104/DC1.

© 2007 by The National Academy of Sciences of the USA

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biology of these systems. Earlier TRNOE studies of weaklybinding peptides derived from PACAP indicate that the peptidehormone adopts a helical structure when bound to the receptor(13). Models of VIP/VPAC1-R based on photoaffinity labelingand homology modeling have been proposed (14) that define thegeneral regions of interaction between the hormone and recep-tor. Recently, the solution structure of the N-terminal ECdomain of the related corticotropin-releasing factor (CRF)receptor has been described (15). Although this study proposeda putative peptide-binding surface consistent with chemical shiftperturbations, no structural data on the protein-peptide complexwas obtained. Thus, additional structural data on the peptidereceptor complex is necessary to provide a better understandingof the interactions that stabilize complex formation.

In this article, we report our results on the structure deter-mination of the N-terminal EC domain of the hPAC1-RSreceptor in complex with the PACAP (6�–38�) peptide antago-nist. The structure was used to define the binding interfacebetween PACAP and the N-terminal EC domain, and a muta-tional analysis of the receptor and PACAP peptides was used todetermine the energetic contribution from these residues tocomplex formation. A comparison of the binding affinity for thefull-length receptor and the isolated N-terminal EC domainallowed a separation of the binding interactions contributedfrom the N-terminal EC domain and other extracellular regionsof the receptor.

Results and DiscussionStructural Description. The structure of the hPAC1-RS N-terminalEC-domain (residues 21–122, C25G)/PACAP(6�–38�) complexwas determined by using NMR-derived distance, residual dipolarcoupling, and torsion angle restraints. (Throughout the text,peptide residues are always designated by using an apostrophewith the numbering of full length PACAP.) Fig. 1A depicts asuperposition of the C� trace of 10 low energy structures.Residues 25–120 are well defined except for the loop between�3-�4. For PACAP, the N-terminal residues 6�–18� are lessdefined than the C terminus of the peptide. A summary of thestructural statistics is given in supporting information (SI) Table1. Analysis of the average-minimized structure with the programPROCHECK showed that 67% of the residues for the N-terminal EC domain of hPAC1-RS lie in the most-favored regionof the Ramachandran plot, whereas an additional 33% are inallowed regions.

A secondary structure representation (16) of the averageminimized NMR structure of the N-terminal EC domain of thehPAC1-RS /PACAP complex is shown in Fig. 1B. The structureof the N-terminal EC domain of PAC1-RS consists of anN-terminal helix and four �-strands forming two antiparallelsheets. Three disulfide bonds lock these secondary structureelements together. The helix at the beginning of the domain isconnected to �2 by a disulfide linkage between C34 and C63.Two antiparallel sheets are connected by a disulfide bondbetween C54 (just before the first strand) and C97, which is ina loop after strand �4. After �4 is an extended strand that isterminated by cis P107, leading to the final disulfide bondbetween C77- C113. The buried core of the protein contains theconserved tryptophans (W64 and W102) and aspartate residue(D59) that are characteristic of family members. The trypto-phans span the region between the two antiparallel �-sheets,whereas D59 is in position to form a salt bridge to R95, whichis also highly conserved. Mutation of these residues has beenreported to diminish peptide binding (3, 5). From the structure,it is likely that these mutations cause a disruption of the corestructure that leads to misfolded or less stable N-terminal ECdomains, which would affect peptide binding.

The affinity of PACAP 6�–38� for the soluble N-terminal ECdomain of hPAC1-RS is 350 nM. Under the experimental

conditions used here, the protein is fully bound and is in slowexchange on the NMR time scale, resulting in only one set ofpeaks for the bound conformation. The protein resonances thatshift upon peptide binding are localized to one face of theprotein, suggesting that only limited structural changes arenecessary to accommodate peptide binding.

Free in solution, PACAP 6�–38� exhibits no detectable stablesecondary structure as indicated by the low spectral dispersionobserved in the 1H-15N HSQC spectrum (SI Fig. 6). However,upon binding to hPAC1-RS, the dispersion of the peptide amideresonances increases, indicative of the formation of definedstructure. Our NMR structural studies of the complex indicatethat residues 10�-30� of PACAP are helical when bound to thehPAC1-RS N-terminal EC domain. PACAP residues 29�–34�show Nuclear Overhauser effect (NOE) contacts to Y118, E117,and E119 of the protein, which defines the orientation of the Cterminus of the peptide. Residues 35�–38� at the very C terminusof PACAP are disordered based on their line widths and nearrandom coil chemical shifts. The N terminus of PACAP crosses�3 and contacts L74. To make this contact, the PACAP helixbends at A18�. This bend allows the N terminus of PACAP(residues 10�–17�) to run over �3–�4, whereas the C terminus ofthe peptide (residues 26�–34�) goes over the disulfide bond(C77–C113) and ends near the C terminus of the N-terminal ECdomain. This binding mode places PACAP and the helix of theN-terminal EC domain on opposite sides of the central �-sheetcore of the protein. PACAP, when bound, has good chargecomplementarity to the N-terminal EC domain (Fig. 3).

Multiple splice variants have been identified for PAC1-R withdifferent affinities for PACAP-27 and PACAP-38 as well asdifferential coupling to adenylate cyclase and phospholipase Cpathways. There are three splice variants in the N-terminal ECdomain, PAC1-Rnull (wild type), PAC1-RS (21-aa deletion) andPAC1-Rvs (45-aa deletion). hPAC1-Rnull contains an additional21 aa not present in the other proteins, as shown in the sequencealignment in Fig. 2A. This insert would be located in the loopbetween �3 and �4 and could be accommodated without dis-turbing the core structure of the N-terminal EC domain. Theseadditional residues would most likely be involved in the bindingof the C-terminal part of the PACAP peptide. The presence ofthis 21-aa insert could thus influence receptor selectivity forPACAP-27 and PACAP-38 as well as phospholipase C signalingpathways.

Comparison with Mouse CRF-R2� Receptor and VPAC1/VIP. Recently,the structure of the N-terminal EC domain of the mouseCRF-R2� receptor has been reported (15). This structure has a

Fig. 1. Solution structure of hPAC1-Rs/PACAP complex. (A) Superposition ofthe C� trace of 10 low-energy NMR-derived structures. Residues 25–120 ofhPAC1-RS and residues 10�–30� of the PACAP peptide were used in the super-position. The atomic rmsd for the backbone atoms is 1.00 � 0.17 Å and 1.40 �0.26 Å for all heavy atoms in the complex. Residues 27–118 of hPAC1-RS andresidues 6�–36� of PACAP are displayed. (B) Ribbons depiction of the averageminimized structure. Secondary structure elements and disulfides of thehPAC1-RS N-terminal EC domains are labeled. The ribbon for PACAP is coloredcyan.

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similar �-sheet core, but hPAC1-RS has an additional N-terminalhelix and a short extended strand after �4. An N-terminal helixmay not be observed in CRF-R2� because the domain has fivefewer residues before the start of the first �-sheet than hPAC1-RS, and this could destabilize any helical structure in CRF-R2�.When the two structures are compared, it can be seen thatresonances that shift upon binding of the peptide Astressin B tothe N-terminal EC domain of CRF-R2� map to a similar but notidentical region as we find for PACAP binding to hPAC1-RS(SI Fig. 7).

A model for VIP binding to VPAC1-R has also been reportedbased on photolabeling of the receptor and NMR studies of theVIP peptide in solution (14). The photolabeling studies indicatedthat substitution of benzoylbenzoyl-L-Lys at positions Y22� andN24� of VIP modified residues G116 and C122, respectively, ofthe N-terminal EC domain of VPAC1-R (14). This is consistentwith our structure, where the corresponding residues Y22� andA24� of PACAP are close to the corresponding residues F106and C113 in hPAC1-RS (Fig. 2). In addition, they found thatphotolabeling F6� of VIP modified residue D107 of VPAC1-R(which is homologous to residue E99 of hPAC1-Rs). To satisfythis constraint, they modeled the peptide as running parallel with�3–�4. However, in our structure, residues 6�–10� of PACAPruns across (instead of parallel to) �3–�4, causing F6� to befarther away from E99.

Mutational Studies. Based on our structural data, mutationalstudies on the PACAP peptide were carried out to quantify theimportance of individual residues for binding to the receptor(Fig. 3A). PACAP truncations (ending at 36�, 34�, 32�, and 30�)had only moderate effects (i.e., �3-fold) on affinity, consistentwith the observation that these residues are unstructured in thecomplex. However, the PACAP (1�–28�) truncation resulted ina �10-fold loss in affinity. This decrease in affinity is consistentwith the loss of the contacts between residues 29�–32� of PACAPand E117-Y118 of the protein. From the structure, the centralPACAP hydrophobic residues make significant contacts to theprotein. Consistent with this, we found that single point muta-tions of the central hydrophobic residues V19�G, Y22�A, V26�G,and L27�A all had a significant (�20-fold) effect on affinity for

binding to the N-terminal EC domain. These data indicate anextensive hydrophobic interaction between the N-terminal ECdomain and residues 18�–27� of PACAP. Mutations of residuesin the N-terminal part of PACAP (e.g., Y10�A and R14�A) hada negligible effect on binding to the isolated N-terminalEC domain.

Mutations were also made in the soluble N-terminal ECdomain and evaluated for binding to the PACAP peptide (Fig.3B). The Y118A mutant had the greatest (�10-fold) effect onpeptide binding. Mutation of the adjacent residue E117R ex-hibited an �3-fold loss in affinity for the peptide. The otherprotein mutants had smaller effects on affinity. From thesequence alignment in Fig. 2 A, it can be seen that the contactregion, residues 116–120 (DEYES), results in an acidic patch onhPAC1-RS that complements the basic end of the peptide.However, in VPAC1-R, the corresponding residues areDDKAA, with a basic residue replacing the tyrosine in hPAC1-RS. This lysine residue would likely reduce the affinity ofPACAP-38 to VPAC1-R if its binding mode is similar to what wehave found for hPAC1-RS. This is consistent with the reportedaffinity of peptides binding to human VPAC1-R, whereVIP�PACAP-27 � PACAP-38 (5). These data support the roleof the interaction between the C terminus of the peptide(residues 28�–32�) and the C terminus of the N-terminal ECdomain in defining the high affinity and specificity of hormonebinding to hPAC1-RS. These data also help explain the selec-tivity of PAC1-RS for PACAP-38 over VIP and PACAP-27.

Comparison of the Isolated N-Terminal Domain and the Full-LengthReceptor. The PACAP peptides were also evaluated for bindingto the full-length receptor in a radioligand binding assay. Con-sistent with earlier reports on related receptors (17–19), thepeptide hormone PACAP binds with �1,000-fold higher affinityto the full-length receptor when compared with the solubleN-terminal EC domain. Part of this increased affinity for thefull-length receptor is likely due to the influence of the mem-brane environment (18), where the peptide could preform intoa helical structure (13) and diffuse in two dimensions to interactwith the receptor. Nonetheless, despite the absolute differencein affinity, there is a good correlation observed for mutant

Fig. 2. Sequence alignment of the N-terminal EC domain of several related class II GPCRs (A) and their corresponding peptide hormones (B). The secondarystructure for hPAC1-RS is indicated above the sequence. The results from the mutational data are summarized by using open, half-filled, and filled circles toindicate no effect, 3- to 5-fold, and �5-fold decrease in binding, respectively, upon mutation of the residue.

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peptide binding to both the full-length receptor and the N-terminal EC domain (Fig. 4 and SI Table 2), suggesting that thepeptide-binding mode observed for the isolated N-terminal ECdomain is conserved in the full receptor. In particular, mutationand truncation of residues in the C-terminal part of the peptide(Y22�–K38�) have the same relative effect on binding to the fullreceptor and the isolated N-terminal EC domain, stronglyindicating that these residues of the peptide hormone interactalmost exclusively with the N-terminal EC domain of hPAC1-RSeven in the membrane environment. In contrast, several residuesin the central region of the peptide (Y10�A, R14�A, and K21�E)exhibit a moderately higher effect on binding to the full receptorrelative to the N-terminal EC domain (see Fig. 4). In ourstructure, these residues are aligned on the exposed face of thehelical peptide when complexed to the N-terminal EC domain(see Fig. 3A) and thus are accessible for making additionalbinding interactions with other parts of the full-length receptor.

Previous binding studies of PACAP peptides have delineatedthe critical role of the N-terminal residues 1�–10� in receptorbinding and activation (20). However, removal of the first fiveresidues (1�–5�) of PACAP had no effect on binding to theN-terminal EC domain (Fig. 3A). In addition, whereas intrapep-tide constraints indicated that residues 6�–9� are in a helical

conformation when bound to the N-terminal EC domain, noNOE contacts could be identified between these residues and theN-terminal EC domain. These data suggest that the N-terminalresidues of PACAP (e.g., 1�–9�) interact with other parts of thereceptor outside of the N-terminal EC domain, such as theextracellular loops or the transmembrane helices of the receptor.

Taken together, the structural, mutational, and binding dataare consistent with a model for peptide hormone binding to classII GPCRs shown schematically in Fig. 5. This model has thePACAP peptide complexed to the N-terminal EC domain andlying along the extracellular region of the transmembrane do-main. This orientation allows PACAP residues Y10�, R14�, andK21�, which we have shown are important for binding tofull-length hPAC1-RS (Fig. 4), to contact parts of the receptoroutside of the N-terminal EC domain, such as the first EC loop(Fig. 5 colored green). Mutagenesis and photolabeling studies onVIP and PTH binding to their respective receptors have indi-cated the importance of the residues in the first EC loop for

Fig. 3. Charge density surface for hPAC1-RS (A) and PACAP (B). Side chains of selected residues are rendered and labeled. PACAP mutations used the peptide(6�–38�) as template. The binding data for hPAC1-RS/PACAP complex are given in nM units, with the standard deviation of triplicate measurements in parentheses.

Fig. 4. A comparison of ��G for PACAP mutant binding to the isolatedN-terminal domain and the full hPAC1-RS receptor. Standard Gibbs free energy�G is calculated as �G � RTlnKI, where R is the gas constant (1.987 cal � K1 �

mol1), T is the absolute temperature (298 K), and ��G � �G (wild type) �G(mutant).

Fig. 5. Model of PACAP binding to full-length PAC1-R. The N-terminal ECdomain of PAC1-R is shown in pink, and the transmembrane segment of thereceptor (modeled by using the rhodopsin structure, PDB ID code 1HZX) is shownin gray. The PACAP peptide used in this study is colored in cyan, with the residuesthat caused a larger change in binding to the full-length receptor (see Fig. 4)rendered and labeled. Shown in yellow is the structure of the receptor-boundPACAP-21 (13) (PDB code 1GEA) superimposed onto PACAP (6�–38�). The extra-cellular loop between TM helices 2–3 of rhodopsin is colored green.

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hormone binding and receptor activation (5, 21–23). The orien-tation of PACAP in this model is different from that proposedfor Astressin B binding to the N-terminal EC domain of CRF-R2� based on chemical shift perturbations and electrostaticconsiderations, in which the peptide hormone was placed per-pendicular to the membrane surface (15). Superposition of thereceptor-bound structure of PACAP-21 (13) onto the model inFig. 5 places the N-terminal residues of PACAP near the core ofthe helical bundle of the receptor, consistent with the roleof these residues in receptor activation. The requirement forboth the N-terminal and central residues of the peptide tocontact regions of the receptor outside of the N-terminal ECdomain positions the C terminus of the hormone distant fromthe transmembrane domain, in agreement with the structuraland mutational data described in this work.

ConclusionsThe N-terminal EC domains of type II GPCR hormonereceptors have high sequence homology and likely adoptsimilar binding modes for their peptide hormone ligands. Ourstructural and mutational studies have identified hormoneresidues that are critical for binding to the N-terminal ECdomain of PAC1-RS. These data aid in the understanding ofhow receptors differentiate between peptides of very similarsequence and may help in the design of specific antagoniststhat can be used to understand the activities of the receptorsubtypes and ultimately lead to therapeutics for the treatmentof a variety of human diseases.

Materials and MethodsProtein Preparation. The expression of the N-terminal EC domainof hPAC1-RS (human short variant with 21-aa deletion) residues21–122 with a C25G point mutation was accomplished by usinga thioredoxin fusion protein, followed by a His6 tag. The C25Gmutation was chosen because it has similar binding affinity toPACAP38 as the wild-type protein (Fig. 3B) and improvedstability. The protein was expressed in E. coli BL21(DE3) cellsand grown in M9 media. Uniformly 15N, 13C-labeled sampleswere prepared with media containing 15NH4Cl and [U-13C]glu-cose. Deuterated samples were grown by using D2O and[U-2H, 13C]glucose. Soluble protein was purified by Ni2-affinitychromatography, followed by thrombin cleavage and a secondNi2-affinity column to remove the fusion protein and His6 tag.The protein was further purified with a gel filtration column(Superdex-75). Mutants were prepared by using the QuikChangesite-directed mutagenesis kit (Stratagene, La Jolla, CA) andpurified as described above. All protein mutants were checkedfor proper folding by using [1H, 13C]-HSQC spectra (24).

Labeled PACAP (6�–38�) was expressed as a thioredoxinfusion protein in E. coli BL21(DE3) cells. Uniformly 15N,13C-labeled samples were prepared with media containing15NH4Cl and [U-13C]glucose. The fusion protein and His6-tagwere cleaved with enterokinase, giving the native N terminus forthe peptide. The enterokinase enzyme was then removed with acommercial kit (Invitrogen, Carlsbad, CA). Peptides were pu-rified by using HPLC. Unlabeled peptides were prepared in-house by using standard peptide chemistry on a peptide synthe-sizer and purified by using HPLC to �95% purity.

For structural studies, peptide was added at a slight excess tothe protein, followed by removal of excess peptide by using a gelfiltration (Superdex-75) column. Three samples were preparedfor structural studies. One sample contained uniformly 15N, 13C,1H-labeled protein and unlabeled (i.e., 14N, 12C, 1H) peptide. Thesecond sample contained 15N, 13C, 1H-labeled peptide andunlabeled protein. The final sample contained 15N, 13C, 1H-labeled peptide and 2H-labeled protein.

NMR Spectroscopy. NMR samples contained 0.5–1.0 mM hPAC1-RS/PACAP complex in 50 mM phosphate (pH 7.0) and 100 mMammonium sulfate. All NMR experiments were acquired at 298K on a DRX500 or DRX800 NMR spectrometer (Bruker,Billerica, MA). Backbone 1H, 13C, and 15N resonance and sidechain assignments were obtained with samples containing eitherlabeled protein/unlabeled peptide or deuterated protein/labeledpeptide. Triple-resonance experiments [HNCA, HN(CO)CA,HN(CA)CB, HN(COCA)CB, HNCO, and HN(CA)CO] (25, 26)were obtained for both samples. The side chain 1H and 13C NMRsignals were assigned from the two samples by using H(CC-CO)NH, (H)C(C-CO)NH (27), or HCCH TOCSY experiments.Stereospecific assignments for a total of nine valine and leucinemethyl groups of the N-terminal EC domain of hPAC1-RS wereobtained from an analysis of the 13C–13C coupling patternsobserved for biosynthetically directed, fractionally 13C-labeledhPAC1-RS (28). NOE distance restraints were obtained fromthree-dimensional 15N- and 13C-edited NOESY or three-dimensional 12C filtered 13C-edited NOESY spectra acquiredwith a mixing time of 80 ms (29, 30). To simplify analysis of theNOE experiments, these experiments were repeated for both thelabeled protein/unlabeled peptide and the unlabeled protein/labeled peptide.

Residual Dipolar Couplings (RDC) (31) were measured byusing a 0.5 mM 15N, 13C PACAP/15N-labeled hPAC1-RS com-plex. Pf1-phage (ASLA Biotech, Riga, Latvia) was added to aconcentration of 25 mg/ml (32). Dipolar couplings were mea-sured from coupled 15N or 13C HSQC experiments.

Structure Calculations. Initial structures of the hPAC1-RS (resi-dues 21–122,C25G)/PACAP complex were calculated by using asimulated annealing protocol with the program XPLORNIH(33). Three disulfide bonds (C34–C63), (C54–C97), and (C77–C113) were included in the calculations based on observedNOEs between the H� protons of the two cysteines and otherNOEs that were consistent with this disulfide-formation pattern.The final structures were calculated in CNX (34) by using aHADDOCK-type protocol (35) consisting of rigid body dockingof hPAC1-RS(21–122,C25G) and PACAP(6�–38�) starting fromour initial structures of the complex and refined by using the fullNMR restraints, followed by high-temperature (1,000 K) simu-lated annealing and molecular dynamics (2 ns, 300 K) usingexplicit water solvation and full electrostatics. Structures of thecomplex were calculated by using 1,023 intraprotein, 293 in-trapeptide, 46 protein–peptide NOE constraints, 23 RDC, andthree intrapeptide hydrogen bond restraints derived from ananalysis of amide exchange rates and consistency with observedsecondary structure. Because of peptide resonance degeneracy,several ambiguous NOE constraints were also used in therefinement. Torsion angle restraints, � and �, were generatedfrom an analysis of C�, C�, and H� chemical shifts by using theTALOS program (36). Forty-eight protein and 15 peptidetorsional restraints were used from this analysis. A square-wellpotential (FNOE � 50 kcal mol1) was used to constrain NOE-derived distances. Final structures contained no dihedral-angleviolations �5° and no NOE violations �0.2 Å.

Peptide Binding. Peptide binding to the N-terminal EC domain ofhPAC1-RS was determined by using a fluorescence polarizationanisotropy (FPA) competition assay. Fluorescence polarizationmeasurements were conducted as described (37) on an Analyst96-well plate reader (LJL; Molecular Dynamics, Sunnyvale, CA) byusing an Oregon green (OG)-labeled PACAP(6�–38�, K15-OG)peptide as the probe. Titrations were carried out in a buffercontaining 120 mM sodium phosphate (pH 7.55), 0.01% bovine�-globulin, and 0.1% sodium azide. The protein concentration was600 nM with the probe concentration at 25 nM. Dissociation

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constants were determined from titration curves with in-house-written software by using the analytical expressions of Wang (38).

Radioligand Binding Assay. The iodinated peptide, [125I]PACAP-27 [specific activity 2,200 Ci/mmol (1 Ci � 37 GBq);PerkinElmer Life and Analytical Sciences, Boston, MA] wasused in the binding assay. Crude membranes expressinghPAC1-RS (5 �g per well) were incubated at 37°C for 20 min ina total assay volume of 0.25 ml in assay buffer (5 mM MgCl2,0.5% BSA, and 50 mM Tris�HCl, pH 7.4). For saturationexperiments, membranes were incubated with increasing con-centrations of radioligand (0.01–1 nM). Displacement studiesused 0.2 nM [125I]PACAP-27 with varying concentrations ofunlabeled peptides. Nonspecific binding was defined by 300 nMunlabeled PACAP-38. The assay was terminated by rapid vac-

uum filtration through GF/B filters soaked overnight in 0.5%PEI at 4°C. Filters were washed three times with ice-cold harvestbuffer (0.5 mM EDTA, 0.1% BSA, and 50 mM Tris�HCl, pH 7.4)and radioactivity bound to the filters was measured by TopCount(PerkinElmer). For the radioligand binding assay, the KD of theligand (equilibrium dissociation constant) and Bmax (maximalreceptor density) were analyzed by nonlinear regression of thesaturation binding data. The Ki values were determined from theconcentration inhibition curve by the method of Cheng andPrusoff (39). All values were calculated by using the Prismanalysis package (GraphPad, San Diego, CA).

We thank Jonathan Greer and Tom Holzman for support and usefuldiscussions.

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