Structural Determination of the Vasoactive Intestinal Peptide by Two-Dimensional 'H-NMR Spectroscopy Y. THERIAULT,',* Y. BOULANGER,**' and S. ST-PIERRE3 'Dkpartement de Chimie and *lnstitut de G6nie Biomkdical, Universit6 de Montrbal, C. P. 6128, Succ. A, Montreal, Quebec H3C 3J7, and 'Institut National de la Recherche Scientifique-Sant6, Universiti. du Qubbec, Pointe-Claire, Quebec H9R 1G6, Canada
The structure of the vasoactive intestinal peptide 1-28 in 40% 2,2,2-trifluoroethanolwas investigated by two-dimensional 'H-nmr spectroscopy. All 'H resonances, except the y, 6, and c protons of the lysine residues, could be sequentially assigned. Numerous intraresidual as well as short-range interresidual nuclear Overhauser effect spectroscopy connectivities were observed. Using a variable-target function minimization, a molecular model consisting of two helical stretches involving residues 7-15 and 19-27 connected by a region of undefined structure was calculated. The existence of an undefined structure between residues 16 and 18 confers mobility to the peptide molecule.
INTRODUCTlON The vasoactive intestinal peptide ( V I P ) is a gastrointestinal hormone whose multiple roles include vasodilation; reduction of blood pressure; stimulation of the cardiac response; relaxation of the smooth muscles of the trachea, stomach, and gallbladder; and stimulation of gly~ogenolysis.'~~ Its primary structure, consisting of a linear 28-residue chain, is related to glucagon and secretin? Several conformational studies of the VIP and of a VIP analogue have already been reported using techniques such a s optical rotatory dispersion ( O R D ),4,5 CD,6-9and nmr.6.'.9 In aqueous solution, ORD data were consistent with a structure constituted of 20% helix and 80% random The predominance of the random coil structure in aqueous solution was also confirmed by CD and 'H-nmr m e a s u r e m e n t ~ .In ~'~ the presence of 2,2,2-trifluoroethanol (TFE) or 1,1,1,3,3,3-hexafluoroisopropanol, CD and ORD data are consistent with a mostly helical structure for the VIP mole~ u l e .The ~ helix propagation probability was found to be especially high for residues 13-20.8 Another
Biopalymers. Vol. 31, 459-464 (1991) (~21991 J o h n Wiley & Sons, Inc.
* Present address:Department of Molecular Biology, Research Institute of Scripps Clinic, La Jolla, California 92037. To whom correspondence should be addressed.
element of secondary structure is the prediction of two @-turnsin positions 1-4 and 7-10 by Chou-Fasman calculation^.^^^ An exhaustive CD and two-dimensional nmr study of a VIP analogue ( Ac- [ Lys", Lys 1 4 , Nle 17, Val 26, T h r 28] VIP ) has been reported in methanol /water.g Two helical segments a t residues 9-17 and 23-28 were found in 25% methanol. In 50% methanol, a long helix for residues 8-26 and a type I11 /3-turn for residues 5-8 were observed. We report here the first complete structural determination of native VIP in 40% deuterated T F E ( TFE-d3) solution performed by two-dimensional 'H-nmr spectroscopy. The internuclear distances estimated by nmr allow the calculation of a mostly helical molecular model using a variable-target function minimization.
MATERIALS AND METHODS Human VIP 1-28 was synthesized in the laboratory of Dr. S. St-Pierre (in collaboration with Dr. A. Fournier) or was purchased from Bio-Mega, Inc. (Montreal). TFE-d3and deuterium oxide 100% were obtained from Merck, Sharp & Dohme Isotopes (Montreal). VIP samples for nmr were dissolved in a 60 : 40 mixture ( v / v ) of phosphate buffer ( 3 m M potassium phosphate, 10 m M KC1, p H 7.2, prepared 459
THERIAULT, BOULANGER, AND ST-PIERRE
in HzO or DzO) and TFE-d3. The peptide concentrations in the nmr samples were between 4 and 5 mM. 'H-nmr spectra were recorded on a Bruker AM 500 spectrometer (500 MHz) equipped with digitalphase shifters and an ASPECT 3000 computer ( Biotechnology Research Institute, Montreal). Phase-sensitive correlation spectroscopy ( COSY ) spectra were acquired using the time proportional phase incrementation method." The nuclear Overhauser spectroscopy ( NOESY ) and relayed coherence transfer spectroscopy spectra were recorded in the absolute value mode as previously described.' '-13 The water resonance was suppressed by selective irradiation at all time except during the acquisition. All experiments were carried out at 20°C. The twodimensional nmr spectra were obtained by apodization with a sine-bell window function prior to Fourier transformation. The chemical shifts were referenced to sodium 4,4-dimethyl-4-silapentane-lsulfonate at 0.0 ppm. Spectral connectivities were identified following a previously described notation.14 A variable-target function calculation in dihedral angle space was used to obtain polypeptide conformations compatible with the NOE data. The computational procedure is as described in V6squez and Scheraga ( 1988) l 5 as modified by Ni et al. ( 1989).16 For NOES involving degenerate protons, no pseudoatoms are used nor are the upper bounds adjusted to take into account the ambiguity. Instead, for NOE interactions between nl indistinguishable protons and n2 other indistinguishable protons, an effective distance deffbetween these is calculated: nl
where d: is the interatomic distance that gives rise to a nonbonded interaction energy of 20 kcal/mol for the Empirical Conformational Energy Program for Peptides p0tentia1.l~At the end of the calculation, the four structures with the lowest residual violations of the distance constraints were reminimized with a contact energy of 3 kcal/mol.
RESULTS AND DISCUSSION Sequential Assignments
The phase-sensitive COSY spectrum of VIP in 40% TFE-d3 buffer allowed the identification of the aamide connectivities (Figure 1) of all residues with AMX spin systems as well as those from serine, isoleucine, leucine, threonine, and alanine. A sequential analysis was then performed using the a-amide and amide-amide (Figure 2 ) regions of the NOESY spectrum. These assignments rely on a-amide ( i , i 1) and amide-amide ( i , i 1) connectivities as well as on the identification of the aliphatic spin systems. The superposition of certain resonances (the a-amide NOESY connectivities of alanine 18 and leucine 13, for example) as well as the coincidence of a significant number of amide-amide ( i , i 1) connectivities with the diagonal (1-2; 2-3; 56; 14-15; 15-16; and 21-22) have created compli-
deff= ( 2
2r k = l I=1
where rklis the calculated distance between protons k and 1. This effective distance is used in the penalty function
41'" where upp is the upper bound deduced from the observation of the given NOE. This functional form makes better use of short range NOE information. Twenty-five random starting conformations were generated and subjected to minimization of the variable-target function. A contact energy of 20 kcal/mol was used, i.e., a penalty function of the form (dij - d;)'
if dij < d $
Figure 1. a-Amide region of the phase-sensitive COSY spectrum (500 MHz) of 5 mM VIP 1-28 in a solvent mixture ( 6 : 4 ) of phosphate buffer ( 3 m M potassium phosphate, 10 mMKC1 in H 2 0at pH 7.2) and TFE-d3at 20°C. The intraresidualconnectivities are identified by their oneletter residue code and sequence number.
STRUCTURAL DETERMINATION OF VASOACTIVE INTESTINAL P E P T I D E
Figure 2. Amide-amide region of the NOESY spectrum (500 MHz) of 5 m M VIP 1-28 in a solvent mixture ( 6 : 4 ) of phosphate buffer ( 3 m M potassium phosphate, 10 m M KC1 in H 2 0 a t pH 7.2) and TFE-d3 at 20°C. Connectivities are identified by the sequence numbers of the residues to which the amide protons belong.
cations in the sequential assignment. In addition, very weak COSY and NOESY connectivities have been observed for the asparagine-28 AMX spin system in both the aliphatic region and the amide region because of the coincidence of its a-proton resonance with the irradiated water resonance. In spite of these difficulties, a complete assignment has been realized and the chemical shift values are listed in Table I. lnterspatial Connectivities
A network of systematic "d
(i, i 1)connectivities exists for the VIP peptide (Figure 3 ) . Several correlation peaks are not observed because of the small difference in chemical shift for the connecting nuclei, which leads to a superposition with the diagonal. This is evident from the chemical shift values given in Table I for the following pairs of residues: serine 2 and aspartic acid 3, valine 5 and phenylalanine 6, arginine 14 and lysine 15, lysine 15 and glutamine 16, as well as lysine 2 1 and tyrosine 22. I t must also be noted that no amide-amide interresidual connec-
tivity other than between successive residues has been observed. T h e existence of a defined secondary structure is supported by the observation of d a N ( i, i 2 ) , d a N (i, i 3 ) , and d a N ( i , i 4 ) connectivities (Figure 3 ) . These connectivities are typical of helical structure. They are observed from residue 2 to residue 27, but residues 17 and 18 are not implicated in such connectivities. The lack of d a N connectivities (between nonneighboring residues) in the segment corresponding to residues 17 and 18 suggests a high mobility of the peptide, which might be in fast continuous bending motion in that region, leading to a drastic loss of NOE.
T h e distance constraints used for the calculation of the three-dimensional structure can be derived from Figure 3 where the connectivities with strong, medium, and weak intensities were assigned interproton distances of I 2.5, I 3.5, and I 5 A, respectively,
THERIAULT, BOULANGER, AND ST-PIERRE
Table I Proton Chemical Shifts of VIP (1-28) in 60% Aqueous Phosphate Buffer: 40%TFE-d3 Chemical Shift (ppm) Residue
3.48 3.92 4.02 3.00 3.08 1.44 2.04
3.16 3.23 4.31 2.95 3.06 2.77 2.86 3.00
2.01 2.33 2.18 2.14 2.26 1.61 2.26
Lys-20" Lys-21" Tyr-22
8.09 8.27 8.29
4.09 4.06 4.31
1.98 2.03 3.26 3.30 1.50 2.82 2.93 4.09 1.87
Others C2H 8.69 C4H 7.48
0.86 0.94 Aromatic 7.18 7.30 1.30
Aromatic 7.07 6.83 1.33 1.82 1.62 1.74 1.58 1.44
6 3.25 tNH 7.13 6 0.96 6 3.08 cNH 7.05
1.36 (CH,) 1.05 (CH,) 0.80 (CH,) 1.75
Aromatic 7.09 6.78 6 0.92
6 0.80 6 0.86
a Spin systems for the Lys side chains could be identified but could not be unambiguously connected to the p protons.
STRUCTURAL DETERMINATION OF VASOACTIVE INTESTINAL PEPTIDE
1 2 3 4 5 6 7 8 9 10 111213141516171819202122232425262728 H S D A V F T D N Y T R L R K Q M A V K K Y L N S I L N daN
dYN dm d"
daN 0,i+l) dPN(i,i+l)
42J (i, i+l)
d m (i,i+l) dNa (i,i+l) dNy (i, i+l) daN (i, i+2) daN (i, i+3)
daN (i, i+4)
Figure 3. Intra- and interresidual NOESY connectivities observed for VIP 1-28 in a solvent mixture ( 6 : 4 ) of phosphate buffer ( 3 m M potassium phosphate, 10 m M KCl in H 2 0 at pH 7.2) and TFE-d3 at 20°C. The thick, medium, and narrow lines correspond to intense ( 5 2.5 A ) , medium ( 5 3.5 A ) , and weak ( 5 5 A ) connectivities, respectively.
on the basis of peak volume measurements. The stereo view of the structure with the lowest residual violations of the distance constraints after the variable-target function minimization is shown in Figure 4.This structure displays no violation greater than 0.5 A. The main structural feature is the presence of two helical stretches from residues 7-15 and residues 19-27, but the orientation of the helical segments with respect to each other cannot be predicted due to the absence of long-range connectivities between the two segments. The two segments are therefore separately displayed in Figure 4.The last four calculated structures had nearly identical helical segments and therefore only the optimal one is displayed. All J H N coupling a constants in these regions were below 6 Hz, in agreement with a helical structure. The random orientation of the two segments relative to each other indicates that while the secondary structure is well defined, the tertiary fold of this peptide is not uniquely defined. The structure OfpI' is found to be from the analysis of the two-dimensional nmr data, in agreement with CD and ORD results.4-7 These results are not in agreement with the calculation of a maximal helix propagation probability between
Figure 4. Stereo structures of two segments of VIP 128 calculated by the variable-target function method using the NOESY connectivity data: ( A ) residues 1-17 and ( B ) residues 18-28. Helix structure is calculated between residues 7 and 15, and between residues 19 and 27. The maximal distance violations were less than 0.5 A.
THERIAULT, BOULANGER, AND ST-PIERRE
residues 13and 20 for VIP in sodium dodecyl sulfate on the basis of CD results.s Under our conditions, this central region is the least defined. The difference could be attributable to the different medium used in the two experiments. Our results are qualitatively similar to the results of the nmr study of a VIP analogue in 25% methanol /water where two helical segments have been observed.' The location of the segments is however slightly different: 9-17 and 23-28 for the VIP analogue compared to 7-15 and 19-27 for VIP. Our structure is more different from the reported structure for the VIP analogue in 50% methanol/water. In that case, a long a-helix between residues 8 and 26 is obtained in addition to a type I11 /3-turn between residues 5 and 8.' The differences between the two studies can be explained by the fact that the replacement of the arginines in positions 12 and 14 by lysines enhances the helical character, and by the fact that different solvents were used. The structures of the functionally related secretin and glucagon are more irregular than the VIP structure."," Each peptide displays short helical and random coil segments when studied in 40% trifluoromethanol ( secretin ) and perdeuterated dodecylphosphocholine micelles (glucagon). It should be mentioned that more than 70% of the residues are different between VIP and these two peptides. It is interesting to note that in many peptides of that size, helical segments linked by a region of undefined structure have been reported. This has been the case of melittin,20of secretin," and of the growth hormone releasing f a c t ~ r . ' ~ , ~ ' The variable-target function calculation was chosen because of its following advantages: (1)its sampling properties are better than in the case of distance-geometry algorithms, ( 2 ) the sum over the sixth power of the distances makes better use of sequential NOES, and ( 3 ) the calculations are less time-consuming.'6 The minimal violations obtained are relatively small and the model generated must represent accurately the VIP structure constituted of two helix stretches linked by a random coil peptidic chain portion that confers mobility to the molecule. In less polar media or in more restricted environments, as is expected to be the case at the receptor binding site, the central portion of the molecule might also adopt a better defined structure. The authors are especially grateful to Dr. Enrico Purisima from the Biotechnology Research Institute, National Research Council, Montreal, for performing the structural calculations. They would like to thank Drs. Andrew Storer and Irena Ekiel for giving access to the nmr spectrometer at the Biotechnology Research Institute. The participation
of Dr. Alain Fournier, Institut National de la Recherche Scientifique-SantC, Montreal, in the synthesis of the VIP peptide is also acknowledged. This work was supported by grants from the Natural Science and Engineering Research Council of Canada, and from the Heart Foundations of Canada. YT is the recipient of a postgraduate scholarship from the Fonds pour la Formation de Chercheurs et 1'Aide & la Recherche. YB is the recipient of a researcher fellowship from the Fonds de la Recherche en SantC du QuBbec.
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versitB de Sherbrooke, Sherbrooke, QuBbec, Canada. 7. Fournier, A., Saunders, J. K., Boulanger, Y. & StPierre, S. (1988) Ann. N Y Acad. Sci. 527, 51-67. 8. Robinson, R. M., Blakeney, E. W., Jr. & Mattice, W. L. (1982) Biopolymers 2 1 , 1217-1228. 9. Fry, D. C., Madison, V. S., Bolin, D. R., Greeley, D. N., Toome, V. & Wegrzynski, B. B. (1989) Biochemistry 2 8 , 2399-2409. 10. Marion, D. & Wuthrich, K. (1983) Biochem. Biophys. Res. Commun. 113,967-974. 11. Eich, G., Bodenhausen, G. & Ernst, R. R. (1982) J. Am. Chem. SOC.1 0 4 , 3731-3732. 12. States, D. J., Haberkorn, R. A. & Ruben, D. J. ( 1982) J . Magn. Reson. 4 0 , 286-292. 13. ThBriault, Y., Boulanger, Y. & Saunders, J. K. (1989) Biopolymers 27,1897-1904. 14. Wuthrich, K. W., Billeter, M. & Braun, W. (1984) J . Mol. Biol. 180, 715-740. 15. VBsquez, M. & Scheraga, H. A. (1988) J. Biomol. Struct. Dynam. 5 , 757-784. 16. Ni, F., Meinwald, Y. C., Vbsquez, M. & Scheraga, H. A. (1989) Biochemistry 28,3094-3105. 17. Momany, F. A., McGuire, R. F., Burgess, A. W. & Scheraga, H. A. (1975) J . Phys. Chem. 79,2361-2381. 18. Braun, W., Wider, G., Lee, K. H. & Wuthrich, K. (1983) J . Mol. Biol. 169,921-948. 19. Clore, G. M., Nilges, M., Briinger, A. & Gronenborn, A. M. (1988) Eur. J. Biochem. 171,479-484. 20. Bazzo, R., Tappin, M. J., Pastore, A., Harvey, T. S., Carver, J. A. & Campbell, I. D. (1988) Eur. J . Biochem. 173, 139-146. 0 1 01. Clore, G. M., Martin, S. R. & Gronenborn, A. M. (1986) J . Mol. Biol. 191,553-561.
Received April 26, 1990 Accepted January 18, 1991