Proc. Natl. Acad. Sci. USAVol. 91, pp. 2206-2210, March 1994Biochemistry

Solution structure of a trefoil-motif-containing cell growth factor,porcine spasmolytic protein

(ref potein/protein fold/receptor binding/NM spetropy)

MARK D. CARRtt, CHRISTOPHER J. BAUER§, MICHAEL J. GRADWELLt, AND JAMES FEENEYttLaboratory of Molecular Structure and Medical Research Council Biomedical NMR Centre, National Institute for Medical Research, The Ridgeway, MillHill, London NW7 1AA, England

Communicated by Arnold Burgen, November 29, 1993

ABSTRACT The porcine spasmolytic protein (pSP) is a106-residue cell growth factor that typifies a family of eukary-otic proteins that contain at least one copy of an =40-aminoacid protein domain known as the trefoil motif. In fact, pSPcontains two highly hmlgu trefoil domais. We havedetermined the complete three-dimensional solution structureof pSP by uing a combiation of two- and three-dimensional1HNMR spectroscopy and distance geometry calculation. pSPis a relatively elongated molecule, consisting of two compactglobular domains joined via a small interface. The protein'stwo trefoil domains adopt the same tertiary structure andcontain a core C-terminal two-stranded atiparallel P-sheet,preceded by a 6-residue helix that packs against the N-terminal1-strand. The remainder of the protein backbone is taken upby two short loops that lie on either side of the «-hairpin andare linked by an extended region that wraps around theC-terminal 3-strand. The topology of the protein backboneobserved for the trefoil doma in pSP represents an unusualpolypeptide fold. A strking feature of both trefoil domains Isa surface patch formed from five conserved residues that haveno obvious ructural role. The two patches are located at thefar ends of the protein molecule, and we propose that theseresidues form at least part of the receptor binding site, or sites,on pSP.

Members of an important family of eukaryotic cell growthfactors are characterized by the presence of at least one copyof an -40-amino acid protein domain, termed the trefoilmotif, that contains three conserved disulfide bonds (1-4).The family is typified by porcine spasmolytic protein (pSP)and human breast-cancer-associated protein pS2 (1-4). Re-cently, trefoil domains have been identified in a number oflarge multidomain proteins, such as frog integumentary mu-cins (5) and the major rabbit zona pellucida protein Zpx (6),indicating an additional more general role as a shuffledmodule, probably involved in mediating protein-protein in-teractions.The porcine (pSP), murine, and human (hSP) spasmolytic

proteins are monomeric 106-residue cell growth factors thatcontain two highly homologous trefoil domains (nearly 50%6'amino acid identity), suggesting that the proteins arosethrough gene duplication (Fig. 1) (1-4, 6). The sequences ofthe trefoil domains in the spasmolytic proteins are veryclosely related to the single trefoil motifpresent in the humanbreast-cancer-associated protein pS2 (2). pS2 is a 60-residuecell growth factor of significant clinical and pharmaceuticalinterest, since it is expressed at fairly high levels in estrogen-dependent breast tumor cells, whereas there is no significantexpression in normal human breast tissue (2, 7-10). Recentstudies of pSP, hSP, and pS2 suggest that the proteins playa major role in the repair and7 maintenance of endodermal

tissues, for example, in the healing of ulcers in the gut,probably in conjunction with epidermal growth factor (3, 11).Evidence from work on pSP and pS2 suggests that the trefoilgrowth factors exert their effects via binding to a family ofcell-surface receptors leading to changes in intracellularcAMP levels (2-4, 12, 13).

Clearly, it is important to understand the precise role andmechanism of action of this family of eukaryotic cell growthfactors, a task that will require detailed knowledge of thestructures of trefoil proteins. Recently, we reported theresults of two (2D)- and three (3D)-dimensional 1H NMR-based structural studies of pSP that allowed us to obtainnearly complete sequence-specific 1H resonance assignmentsfor the protein and to determine its secondary structure insolution (1). The observed patterns of sequential (i, i + 1),medium-range (i, i < 5), and long-range (i, i > 4) backbone tobackbone nuclear Overhauser effects (NOEs) clearly showedthat the protein's two trefoil domains adopt essentially thesame secondary structure in solution (summarized in Fig. 1).The main feature ofeach domain is a 6-residue helix followedby a short antiparallel 3-sheet formed from two 5-aminoacid strands (1). This supersecondary structure clearlyidentified the trefoil motif as an unusual class of growthfactor-associated module (1), distinct from other types ofhighly disulfide cross-linked domains, such as those foundin epidermal growth factor and insulin-like growth factor I(2). In this communication we report the complete 1HNMR-based 3D solution structure ofpSP, which representsa significant advance toward the goal of obtaining a detailedunderstanding of the mechanism of action of trefoil growthfactors.

MATERIALS AND METHODSpS8 Samples. Purified pSP was obtained as a gift from Lars

Thim (Novo-Nordisk, Copenhagen) (14). The NMR spectrawere recorded from 0.6-ml samples of 5 mM pSP (pH* 6.2)dissolved in either 100% 2H20 or 90% H20/10% 2H20 asappropriate (pH* values refer to the actualpH meter readingsuncorrected for deuterium isotope effects).NMR Measurements. The 1H NMR experiments were

carried out on a Varian Unity 600 spectrometer at 400C. All

Abbreviations: pSP, porcine spasmolytic protein; hSP, human spas-molytic protein; NOE, nuclear Overhauser effect; NOESY, NOEspectroscopy; ROESY, rotating frame NOE spectroscopy; DQF-COSY, double quantum filtered correlation spectroscopy; PE-COSY, primitive exclusive correlation spectroscopy; TOCSY, totalcorrelation spectroscopy; 2D and 3D, two and three dimensional,respectively.*To whom reprint requests should be sent at present address:Laboratory ofMolecular Structure, National Institute for BiologicalStandards and Control, Blanche Lane, South Mimms, Potters Bar,Hertfordshire, EN6 3QG, England.'The atomic coordinates and structure factors have been depositedin the Protein Data Bank, Chemistry Department, BrookhavenNational Laboratory, Upton, NY 11973 (reference 1PCP).

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Turn Domain II

Helix Domain I Turn Domain Il -

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5 10 15 20 25 30 35 40 45 50 55EKPAACR - CSRQDPKNRVNCGFPGITSDQCFTSGCCFDSQVPGVPWCFKP - LPAQESEE

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C--#E P--R- NCGYPGIT-DQC - -KGCCFDSSI--V-WCFFPXDX D XFXXVS EE RN XYSDTV X X YYXX X X QD XX WNNXX H

XX H X X

FIG. 1. Amino acid sequence of pSP shown with the two trefoil motifs aligned (residues 8-49 and 58-98). The locations of the elements ofregular secondary structure are indicated above the protein sequence (1), as is the topology of the NMR-determined disulfide bonds, whichcorresponds to the arrangement proposed by Thim (2). In addition to the disulfide bonds indicated above, the N- and C-terminal regions ofpSPare linked via a disulfide bond between Cys-6 and -104. Below the amino acid sequence of pSP is shown a consensus trefoil domain sequence,derived from a comparison of the 27 known trefoil motifs. The sequences considered were from pSP, hSP, murine spasmolytic protein, pS2,intestinal trefoil factor, Zpx, APEG, xPl, xP2, xP4, and integumentary mucins Al and C1 (5). The residues or groups of residues included inthe consensus trefoil motif are found in at least 50%o of the sequences examined, and those in boldface type are present in >70%6. The symbol# is used to represent hydrophobic residues. The amino acids shown by the NMR studies of pSP to be conserved for structural reasons areindicated by an asterisk. The five conserved residues identified by a diamond form a conserved patch on the surface of both domains I and IIof pSP, at the extreme ends of the protein molecule, and are proposed in this communication to form receptor binding sites.

the 2D and 3D 1H spectra were acquired in a phase-sensitivemode by using the method of States et al. (15). Spectra from2D double-quantum-filtered correlation spectroscopy (DQF-COSY) (16), total correlation spectroscopy (TOCSY) (17,18), and NOE spectroscopy (NOESY) (19, 20) on pSP wereobtained as described (1). In addition, similar acquisition andprocessing parameters were used to obtain 2D primitiveexclusive correlation (PE-COSY) (21) and 35-ms mixing timerotating frame NOE (ROESY) (22) spectra of pSP.The 3D NOESY-TOCSY (23) spectrum was recorded from

a sample of pSP in 90% H20/10%1 2H20 by using a NOEmixing time of 100 ms and a 50-ms spin-lock period. Theexperiment was acquired over 4 days collecting 128 x 128increments, 8 scans per increment, and 1024 points per scan,using a spectral width of 8000 Hz. Water suppression wasachieved by the use of selective on-resonance presaturation.To observe cross peaks involving protons resonating at orvery close to the chemical shift of water, a 60-ms stimulatedcross peaks under bleached alphas sequence was includedafter the presaturation period (24).The 3D experiment was transformed, displayed, and plot-

ted using a Sun Microsystems (Mountain View, CA) SPARC-station 10 model 41 utilizing software written in-house. Toimprove the resolution in the final spectrum, the number ofdata points in F, and F2 was initially doubled using linearprediction (25). The time-domain matrices were then zero-filled to 256 x 512 x 2048, resulting in a spectrum consistingof 128 x 256 x 1024 real points. In addition, mild resolutionenhancement was obtained by applying a 'r/2.5 shifted sine-squared apodization function in all dimensions.

Stereo-Specific Resonance Assinents and Determinationof Dihedral Angles for pSP. DQF-COSY, PE-COSY, andROESY experiments on pSP were used to obtain estimates ofNH-aCH and aCH-,3CH coupling constants and to determinethe relative intensities ofintraresidue NOEs involvingNH oraCH protons. This information was used as input for a stereosearch program, similar to that described by Nilges et al. (26)(A. N. Lane, personal communication), to determine stereo-specific assignments for signals from ,B-methylene or valinemethyl groups and to define allowed ranges for 4 and X1(26-29). The minimum ranges used in the structural calcula-tions were ±30° for 4 and ±20° for X'.

Calculation and Analysis of pSP Structures. Interresidue'H-1H distance constraints were derived from 2D NOESYspectra of pSP recorded with mixing times of 75 and 150 ms(1) and from the 3D NOESY-TOCSY experiment. Based onthe number of exponentially spaced contours observed forpeaks in the 150-ms NOESY spectra, the NOEs were dividedinto five classes, corresponding to upper-distance limits of2.7, 3.3, 4.0, 5.0, and 6.0 A. In all cases, the lower limits forinterproton distances were equal to the sum of the atomicradii. The upper distance limits were chosen by examiningthe number of contours observed for sequential and mediumrange NOEs corresponding to known 'H-1H distances inelements ofregular secondary structure. In addition to NOE-based constraints, the pSP structural calculations includedappropriate upper and lower distance bounds for 7 disulfidebonds (30) and 4 hydrogen bonds (31), identified from theobservation of 4 slowly exchanging amide protons and cross-strand interresidue NOEs, as described (1). The topology ofthe disulfides in pSP was determined by visual analysis of aset of structures calculated using no disulfide constraints.

Structural calculations for pSP were carried out using thedistance geometry program DIANA (32, 33) installed on aSUN SPARCstation 10 model 41. The yield of properlyconverged structures was substantially improved by makinguse of the redundant dihedral angle protocol (33). Standarddistance corrections were added to constraints involvingpseudoatoms (34). Preliminary pSP structures were calcu-lated from a data set containing upper distance limits derivedfrom 718 unambiguous interresidue NOEs. These allowed theassignment of a further 442 NOEs, which together with theinitial NOE-based distance bounds, 364, 49 V1, and disulfideand hydrogen bond constraints, was used to determine thestructures described here. A total of 19 satisfactorily con-verged pSP structures were obtained from 100 random start-ing conformations. The pSP structures were visualized andanalyzed on a Silicon Graphics (Mountain View, CA) IndigoElan using the Biosym Technologies (San Diego) softwarepackage INSIGHTII.

RESULTS AND DISCUSSIONAnalysis of pSP NMR Spectra. The application of a com-

bination of 2D and 3D 1H NMR spectroscopy allowed nearly

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* Sequential a Medium o Long

FL I i [ I

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-.

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FIG. 2. Histogram showing the number and distribution of the interresidue NOEs identified for pSP plotted against residue number.

complete sequence-specific 1H resonance assignments to bedetermined for pSP (1), by using the sequential assignmentstrategy developed by Wuthrich and coworkers (34). Wehave now used intraresidue NOE and 'H-1H coupling con-stant data from ROESY, PE-COSY, and DQF-COSY exper-iments on pSP to extend this process and obtain stereo-specific assignments for 41 of the 63 pairs of nondegenerate13-methylene signals and for the y-methyl resonances from 5of the 7 valines (26-29).Complete analysis of the 2D NOESY and 3D NOESY-

TOCSY spectra of pSP resulted in the identification of 431sequential, 241 medium-range, and 488 long-range interresi-due NOEs, the distribution of which is summarized in Fig. 2.In addition, allowed ranges were determined for 36 and 49X1 angles and constraints were obtained for 4 hydrogen bonds(1) and 7 disulfide bridges.

Calculation of the Structure of pSP. The solution structuresof pSP were calculated using the distance geometry programDIANA (32, 33), and from a total of 100 random startingconformations, 19 satisfactory structures were obtained. Theconverged structures have no residual NOE, disulfide, hy-drogen bond, or van der Waals violations >0.5 A and no angleviolations >6°. The value of the final DIANA target functionis 6.83 ± 1.06 (average ± SD).

Solution Structure of pSP. As illustrated in Fig. 3, pSP is arelatively elongated molecule consisting of two compactglobular domains joined via a small interface. Domain I is theslightly larger containing residues 2-50 and 104-106, anddomain II is formed from amino acids 58-98 and correspondsto a standard trefoil motif. The interface between the two

involves residues 6, 7, 11, and 50 from domain I and 59-61from domain II. Amino acids 51-57 and 99-103, whichcovalently link the two domains, show very few medium- orlong-range NOEs (Fig. 2), consistent with a high degree ofmobility. Superposition of the backbone atoms for residues2-50, 58-98, and 104-106 gives a rms deviation about theaverage coordinates of 1.54 ± 0.45 A for the family of 19 pSPstructures. This is higher than expected for structures basedon >12 NMR constraints per residue (35) and reflects anuncertainty of ±10° in the 125° rotation about the interface,which defines the orientation of domain I to II (Fig. 3). Therelative orientations of the two domains in pSP are deter-mined by 33 interdomain NOEs involving protons fromresidues 6, 7, 11, 50, and 59-62. However, due to thecombined problems of a small domain I-II interface and theuse of relatively conservative NOE-based interproton dis-tance constraints, to allow for spin diffusion, it is impossibleto decide at present whether the uncertainty in the rotation ofdomain I with respect to domain II reflects mobility or simplyinsufficient NMR data.The conformations of the individual trefoil domains are

well defined, with a rms deviation for the backbone atoms of0.81 ± 0.15 A for domain I (residues 2-50 or 8-49 superim-posed) and 0.53 ± 0.12 A for domain II. This is clearlyillustrated by the family of 19 structures shown for domain Iin Fig. 4. The two domains have the same tertiary structureand consist of a core C-terminal two-stranded antiparallel1-sheet, preceded by a 6-residue helix that packs against theN-terminal -strand. The remainder of the backbone is takenup by two defined loops containing residues 13-16 and 61-64

FIG. 3. Backbone conformation of pSP depicted using a ribbon diagram. Domain I (residues 2-50 and 104-106) is shown in red and domainII (residues 58-98) is in light blue. Residues 1, 51-57, and 99-103, which show few nonsequential NOEs and are believed to be mobile, arehighlighted in yellow.

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FIG. 4. Stereoview of the protein backbone for domain I from pSP. The trefoil domain is shown in red as an overlay of residues 2-50 fromthe 19 satisfactorily converged DIANA structures. The locations of the three conserved disulfide bonds are indicated in yellow, as is the positionof the N and C termini of the domain.

(loop 1) and residues 20-25 and 69-74 (loop 2) that lie oneither side of the /3-hairpin and are linked by an extendedregion that wraps around the C-terminal 3-strand (Fig. 4). Acomparison between the average structure for domain I anddomain II gives a rms deviation for the backbone of 1.34 A.It should be noted that the topology ofthe two trefoil domainsin pSP represents an unusual polypeptide fold.The N and C termini of the trefoil module are close

together, consistent with a role as a shuffled domain used toconfer specific functions to a range of multidomain proteins.The src homology 2 and 3 domains are intracellular examplesof such adaptor modules (36, 37).A comparison of the 27 trefoil domain sequences currently

available (6) identifies 31 positions at which there is a strongpreference for a particular amino acid residue or type ofresidue (Fig. 1). From examination of the structure of pSP, itis clear that the majority of the conservation is for structuralreasons, as indicated in Fig. 1. For example, Ile-24/73,Cys-29/78, Phe-36/Tyr-85, Trp-45/94, and Cys46/95 fromdomains I/IH form a hydrophobic core between the central(3sheet and loop-2-helix region. Similarly, Cys-8/58, Gln-11/Met-60, Arg-16/65, Cys-35/84, Val-40/Ile-89, Val-43/92,and Phe-47/96 form a tightly packed core between the

FIG. 5. Space filling view of the putative receptor binding site ondomain II of pSP. The side chains of the five conserved residuesproposed to interact with the receptor are indicated as follows:Tyr-70, green; Thr-74, yellow; Glu-76, orange; Asp-77, red; Arg-81,dark blue.

13-hairpin and loop 1 in domains I/II. For Phe-47 and Phe-96,we observe distinct signals for both the H2/H6 and H3/H5pairs of ring protons (1), which indicates that the aromaticrings are flipping slowly on the NMR time scale. This isentirely consistent with their location in the solution structureof pSP, where they are placed close to the center of thehydrophobic core of domains I and II, respectively.

Strikingly, on both domains ofpSP, there is a cluster offiveconserved residues, corresponding to Tyr-70, Thr-74, Glu-76, Asp-77, and Arg-81 on domain II, which are located on thesurface of loop 2 and the adjacent helix (Figs. 1 and 5) andhave no clear structural role. We propose that these residuesare conserved since they are required for binding to thereceptor for pSP, which suggests that the protein containstwo receptor binding sites located at the far ends of themolecule. Future work on pSP and hSP should focus on usinga combination of site-directed mutagenesis and NMR-basedstructural studies to further define the functional sites on bothproteins.

We thank Dr. A. N. Lane for many useful discussions and forproviding the stereo search program. The NMR spectra were re-corded using facilities at the Medical Research Council BiomedicalNMR Centre, National Institute for Medical Research, London.

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