Int. J. Peptide Protein Res. 40, 1992, 515-523

Solution conformation of endothelin and point mutants by nuclear magnetic resonance spectroscopy DAVID C. DALGARNO*, LEO SLATER, SAMUEL CHACKALAMANNIL and MARY M. SENIOR

Schering-Plough Research Institute, Bloomfild, New Jersey, USA

Received 21 November 1991, accepted for publication 16 May 1992

Two-dimensional NMR techniques were utilized to determine the secondary structural elements of endothelin- 1 (ET-I), a potent vasoconstrictor peptide, and two of its point mutants, Met-7 to Ala-7 (ETM7A). and Asp-8 to Ala-8 (ETD8A) in acetic acid-d3/water solution. Sequence specific NMR assignments were determined for all three peptides, as well as chemical shifts and NOE connectivity patterns. The chemical shifts of ET-1 and ETM7A are identical ( 5 0.05 ppm) except for the site of substitution, whereas marked shift changes were detected between ET-1 and ETD8A. These chemical shift differences imply that the Asp-8 to Ala-8 mutation has induced a conformational change relative to the parent conformation. All three molecules show the same basic nuclear Overhauser effect (NOE) pattern, which suggests that the gross conformation of all three molecules is the same. Small changes in sequential NOE intensities and changes in medium-range NOE patterns indicate that there are subtle conformational differences between ET- 1 and ETD8A. Key words: endothelin; NMR; point mutations

Endothelin- 1 (ET- 1) is a bicyclic, 2 1 amino acid peptide which was initially isolated from porcine endothelial aortic cells (1). ET-1 and related peptides have been shown to have a wide range of effects on both vascular (1-3) and non-vascular tissues (4-15). Of particular interest is the activity of ET-1, which is one of the most potent vasoconstrictors known. In addition to this effect, it is believed to play a role in renal function (9, 16, 17) and the long term regulation of blood pressure (1, 18). Certain features of the ET-1 structure are

* Present address: Ariad Pharmaceuticals, 26 Landsdowne Street, Cambridge, MA 02139, USA. Abbreviations are defined according to the instructions outlined in J. Biol. Chem. (1967) 242, 4501-4508 and are used in the text of the paper. Other abbreviations are: ABI, Applied Biosystems; CF,COOH, trifluoroacetic acid; DQF-COSY, double quantum filtered two-dimensional correlation spectroscopy; ET-l, endothelin-l; ET-3, endothelin-3; FAB, fast atom bombardment mass spectrometry; Fmoc, 9-fluorenylmethoxycarbonyl; hplc, high performance liquid chromatography; NOE, nuclear Overhauser enhancement; NOESY, two-dimensional nuclear Overhauser enhancement spectroscopy; SRTX, sarafotoxins. The abbreviations used for the NOE types [d”. dmN.d m , daN(i,i + 2), dw(i.i + 4), and d&i + 3)] are defined in ref. 53, p. 116.

known to have a profound effect on activity. These include the two disulfide bonds which stabilize the bioactive structure (19, 20) and the length of the carboxy terminus. Loss of the disulfide bonds causes a dramatic decrease in vasoconstrictor activity (21, 22), while shortening the length of the carboxy terminus decreases potency by three orders of magnitude (23). The sequence of ET-1 and the location of the two disulfide bonds are shown in Fig. 1. Endothelin-1 belongs to a family of peptides which includes endothelin-2 (24), endothelin-3 (l), and four known sarafotoxins (SRTXa-d) derived from the snake venom of Attractaspis engaddensis (25, 26). The endothelins and sarafotoxins are remarkable in their sequence and structural homology and have been shown

FIGURE 1 Amino acid sequence of endothelin-1 showing the location of the disulfide bonds.

515

D.C. Dalgarno et al. to bind to receptors associated with the hydrolysis of phosphoinositides (24-33), and with the mobilization of intracellular calcium ion (22, 34). Amino acid sequence plays a role in the structure-function relationship in the ETjSRTX family. The peptides ET-3, SRTX-c, and SRTX-d are the least potent vasoconstrictors of their family. Their decreased potency is believed to be due to a single change of Ser-2 to Thr-2 (23, 35). Comparison of the seven known amino acid sequences of the ETjSRTX family shows that residues 1, 3, 8-11, 15-16, 18, and 20-21 are always conserved. In contrast to these conserved positions amino acids 4, 6, and 7 are found to be the most variable. In this study, the secondary structural characteristics of ET-1 and two point mutants, one with a mutation at a variable position, Met-7 to Ala-7 (ETM7A), and one with a mutation at a conserved position, Asp-8 to Ala-8 (ETD8A),were determined under similar solution conditions using proton nuclear magnetic resonance spectroscopy ('H NMR). Endothelin has already been the subject of several structural NMR studies. Endothelin does not form a stable concentrated solution in water, consequently these studies were performed using alternate solvent systems which include dimethylsulfoxide (36-40), aqueous acetonitrile (41, 42), aqueous acetic acid (43), and aqueous ethylene glycol mixtures (44, 45). In this work, stable concentrated solutions of ET- 1 and the two point mutants were prepared in an aqueous acetic acid solvent. Sequence specific assignments of the exchangeable NH, C H r P resonances, and most of the sidechain protons were made for all three molecules. From qualitative nuclear Overhauser effect (NOE) patterns, the secondary structural characteristics of the three peptides were elucidated. The effect of the point mutations on the chemical shifts and secondary structural characteristics of the three molecules is discussed. MATERIALS AND METHODS Mnterialy

N-cc-Fmoc-S-trityl-cysteine and N-r-Fmoc-tryptophan-Sasrin resin were purchased from Bachem Bioscience, Inc. All other amino acids were purchased as their N-a-Fmoc derivatives from Applied Biosystems, Inc.: alanine, aspartic acid P-t-butyl ester, glutamic acid b-t-butyl ester, im-trityl-histidine, isoleucine, leucine, o-t-butyl-tyrosine, and valine. Trifluoroacetic acid (CFKOOH), piperidine, hydroxybenzotriazole (HOBt) in N-methylpyrrolidone (NMP), dicyclohexylcarbodiimide in N-methylpyrrolidone, acetic anhydride and 4-dimethylaminopyridine in dimethylformamide were also purchased from Applied Biosystems, The Methylene chloride, acetonitrile, and N-methylpyrrolidone were purchased from Baxter (Burdick and Jackson Division). Optima methanol was obtained from Fisher Scientific. All other reagents were obtained from Aldrich, Inc. 516

Peptide synthesis and purfication of ET-I, ETM7A, and E TD8A The peptides were synthesized by solid phase methods (46) using Sasrin resin (47) with N-cc-Fmoc protection and orthogonal acid (CF3COOH) labile protection for the amino acid side chains. Synthesis was carried out on an ABI Model 430A synthesizer. N-methylpyrrolidine/hydroxybenzotriazole cycles with capping and double coupling were used for all amino acids. Deprotection and cleavage from the support were accomplished by treatment for 3 h with a cocktail of scavengers: (ethylmethylsu1fide:anisole: ethanediol/ CF3COOH)/(3:3: 1:93) total volume 10 mL per 100500 mg resin. The resin was then washed with 1mL C F K O O H followed by 20 niL methylene chloride. The eluent was collected, filtered and concentrated to an orange liquid. The crude endothelin was recovered as a precipitate from cold ethyl ether, dried, and then stirred in dilute aqueous ammonia for 3-4 h. This procedure gave two fully oxidized disulfide isomers in a 3: 1 ratio with the major isomer corresponding to the naturally occurring endothelin. Purification of each peptide was achieved using preparative HPLC with a CSreverse phase column (Rainin Dynamax 41.4 mm I D x 25 cm L, 12 pm 300 A) with UV detection at 229 nni and a stepped gradient. Typical conditions utilized (i.e. for ET-1) were 28/29% acetonitrile in water (0.01 7; CF3COOH) for 60 min. The major isomer eluted at approximately 37 min and 29", acetonitrile. The fractions collected from the preparative run were assayed for purity using a CS reverse phase analytical column [Rainin Dynamax 4.6 mm I D x 25 mm L, 5 pm, 300 A). The pure fractions were pooled, concentrated and lyophilized to a white solid. The lyophilized peptides were analyzed by FAB mass spectrometry.**

NMR spectroscopji Acetic acid-d3 was purchased from M S D Isotopes, Montreal, Canada, and used without further purification. NMR solutions containing acetic acid-dl/watcr (40:60/v:v) were prepared by dissolving the lyophilized peptide in 0.6 mL of solvent to yield a concentration of approximately 2-3 mM peptide, pH 2.5. Reported pH values were measured using a microelectrode and are uncorrected for isotope effects. For the hydrogendeuterium exchange studies, the NMR solutions were lyophilized two times from acetic acid-d3/water before dissolving in acetic acid-d4/deuterium oxide (40:60/v:v). All NMR experiments were carried out on a General Electric GN-500 Omega spectrometer. The temperature of the probe was maintained at 25.0" and the

** FAB mass spectrometry was performed on a VG ZAB-SE model ZAB-SE mass spectrometer. FAB-MS for ET-1: [ M H ] + = 2491.4, calc. = 2491.7: for ETM7A: [ M H ] + = 2433.7: calc. = 2431.8; ETDSA: [ M H ] + = 2449.8, C ~ C =. 2447.9.

Endothelin- 1 conformation TABLE 1 Cheniicul sh$s for (ET-1) in acetic acid-k/waier at 25

NH

a-CH

b-CH

8.80 8.16 8.75 7.71 8.45 8.03 7.51 8.21 8.3 1 7.52 8.02 7.89 8.22 8.59 7.94 7.81 8.20 7.69 7.78 7.88

4.40 4.79 5.04 4.39 4.63 4.19 4.47 4.82 3.95 4.22 4.34 3.58 4.30 4.28 4.66 4.53 4.28 4.69 4.12 4.18 4.74

3.33 3.84 2.65; 3.24 3.93; 4.02 3.70; 3.99 1.68 1.94; 2.18 3.28 1.87 2.18 3.20 2.09 3.01 3.24; 3.20 3.00; 3.27 3.31; 3.40 1.65 2.77; 2.91 1.72 1.75 3.21; 3.30

Residue

sample tubes were not spun. The chemical shift of acetic acid-d3 was referenced to external trimethylsilane. Two-dimensional data were collected with a spectral width of 6514 Hz using a block size of 2048 complex points. Phase-sensitive DQF-COSY (48) and NOESY (49) spectra were acquired in hypercomplex mode according to the method of States et al. (50). NOESY spectra were collected with a mixing time of 250 ms. In both the DQF-COSY and NOESY experiments the

YCH

Others

1.60 2.48; 2.64

0.84; 0.92/CH3

1.45 2.55

1.72 6; 3.04e

0.89; 1.02/CH3 6.86 C2,6H; 6.69 C3,5H 7.33 ring 8.57 C2H; 7.29 C4H 0.80/CH,

1.54

1.04; 1.36 1.07; 1.37

0.62/CH3j.0.80jCH36 0.82/CH,,. 0.89/CH36 7.19 C2H; 7.58 C4H; 7.07 C5H; 7.14 C6H 7.40; 9.86 NH

water signal was suppressed by continuous low power irradiation except during the evolution and acquisition periods . Two-dimensional NMR data were transferred to a Sun 4/360 computer via Ethernet for processing using the program FTNMR (5 1). DQF-COSY data sets were apodized in t2 and tl using a 15 O phase shifted sine-bell squared function. NOESY data sets were processed using either a 60" phase shifted sine-bell squared func-

v12:

v12 :

T 0 I

K91!

1

v12:

0 U

E PI pc

Jo

52%

T

c32

.-I

I

8.8

8.0

PPM

1

8.8

8.0

PPM

FIGURE 2 NH and CH alpha fingerprint region of the DQF-COSY spectra of the peptides (a) ET-1, (b) ETM'IA, and (c) ETD8A. Experimental NMR conditions are described in the text. Additional resonances appearing in the spectra arise from the H16 C2H and W21 ring protons.

517

D.C. Dalgarno et al. tions or a quantitation window function (52). All spectral matrices were zero-filled in tl to yield a final matrix size of 2048 by 2048 real points. Amide exchange spectra were recorded immediately after solvent addition and then at regular time intervals. RESULTS Sequence specific 'H NMR assignments were obtained in an acetic acid-d3/water (40:60/v:v) solvent system using sequential assignment methods (53) for ET-1 and the two mutants, ETM7A and ETD8A. Complete sequential amide-amide and amide-alpha NOE connectivities from residue Ser-2 to residue Asp-18 were observed for all three molecules. The fingerprint regions showing the intraresidue CHa-NH couplings from the DQF-COSY spectra for all three molecules are shown in Fig. 2. The corresponding region in the NOESY spectrum for ET-1 is depicted in Fig. 3. As the DQFCOSY spectra of Fig. 2 show, all of the expected 20 NH-CHsl crosspeaks for each molecule were observed. Table 1 summarizes the measured proton chemical shifts for the NH, CHa, CH/?, and most of the sidechain proton resonances of endothelin. The chemical shifts for ETM7A and ETD8A were also tabulated

but are not shown to conserve space. These tables are available as supplementary material. Once the sequential assignments were completed, it was possible to summarize the relative intensities of the sequential NOE crosspeaks, as shown in Fig. 4. Conformational changes induced by point mutations can be assessed by noting differences in NOE crosspeak patterns and relative intensities from the parent to the mutant molecule. Although it was derived for ET-1, Fig. 4 is representative of all three molecules since the same type of sequential NOE patterns was observed for ET-1, ETM7A, and ETD8A. This similarity in the sequential NOE intensity pattern for all three molecules suggests that at this gross level, the solution structures are qualitatively the same in all three peptides. A detailed analysis of this sequential NOE pattern indicates that the fold of ET- 1 can be described as follows. ET- 1 contains two well-defined structural elements. The first of these is a type 1 /?-turn spanning residues Ser-5 to Asp-8, with Leu-6 and Met-7 located at the corncrs of the turn. The presence of this turn is supported by the appearance of strong d" connectivities between Leu-6 and Met-7, and Met-7 and Asp-8, together with dlN(i + 2) connectivities between Ser-5 and Met-7 and Leu-6 and Asp-8. A turn in this region has been ob-

0

II

1-

\I/

"

8

I

I _ _ _ _ ~ ~

8.'5

8.'0

PPM FIGURE 3 NOESY NH and C H alpha fingerprint region of ET-1 showing the sequential NOES.

5 18

--7.5

Endothelin- 1 conformation

C15

dap(lc3)

0-0

0-0 0 LRange

0

0

FIGURE 4 Summary of the sequential NOE connectivities observed for ET-I I

90

.l..,-. r

l

l

i

, l

,

i

8.5

.,

-

.

v

i

I

8.0

~l

-

~l

i

7.5

i

l

I ~

~

7.0

l

I

l

I

8.5

I

l

/

1

1

1

8.0

1

1

'

1

7.5

I

t

I

l

l

I

'

7.0

ppm

served in other NMR studies (43-45). The second conformational element is a helical structure between Lys-9 and His-16. This structure is supported by the appearance of a string of strong d" connectivities from Lys-9 to Leu-17. These NOEs, the presence of d&i + 3) and daN(i + 3) NOEs, and less intense daN NOEs between residues Lys-9 and His-16 indicate the presence of a helical conformation. Our detection of a helical conformation is in agreement with earlier published secondary structural characteristics of ET-1 in aqueous acetonitrile (41,42), 10% aqueous acetic acid solutions (43), and in aqueous ethylene glycol solutions (44,45). The N- and C-termini of the molecule do not fall into well-characterized structural types. At the N-terminus, residues Cys-1 to Cys-3 appear to be in an extended conformation leading to a poorly characterized turn centered at Ser-4. Similarly at the C-terminus, from Leu-17 to Trp-21, there is again an extended conformation, and in addition, the overall intensity of d"

9.0

l

FIGURE 6 Hydrogen-deuterium exchange profile for the NH protons of ETM7A as a function of time in 30;0 aqueous acetic acid solution at 25".

NOE crosspeaks decreases, implying that a substantial increase in segmental mobility has occurred. Our perception that the N-terminus is a mobile part of the endothelin molecule is in agreement with NOE patterns observed in other solvent systems (43-45). The NOE patterns we observe at the C-terminus are also in agreement with some previously published work (42-45). Hydrogen-deuterium exchange studies The lifetimes of the a i d e protons in all three molecules were followed by adding acetic acid-d3/D20 (40:60/ v:v) to samples lyophilized from acetic acid-d3/water solutions. In all three peptides the NH resonances observed at time zero were El0 through C15 supporting the conclusion that a helical segment exists in this region.

,

I

I

PPm

FIGURE 5 Hydrogen-deuterium exchange profile for the NH protons of endothelin as a function of time in a 30% aqueous acetic acid solution at 25".

I

9.0

8.5

8.0

7.5

7.0

ppm

FIGURE 7 Hydrogen-deuterium exchange profile for the NH protons of ETD8A as a function of time in 30pb aqueous acetic acid solution at 25".

519

D.C. Dalgarno et al. DISCUSSION Conformational changes induced by point mutations are reflected in three principal NMR parameters. Conformational changes induce changes in chemical shift values, cause intensity alterations in NOE patterns, and induce coupling constant changes. Comparisons of the chemical shift data indicate that the chemical shifts of ET and ETM7A are very similar ( 0.05 ppm) with the exception of the site of substitution itself, Met-7 to Ala-7. At this site, a 0.20 ppni shift is observed at the alpha position attributable to random coil differences (54). On a chemical shift basis it appears that the conformation of ET-1 and ETM7A are identical. This is

E a

a

-0.2' 1

"

3

"

5

"

7

"

"

9

"

1 1

Residue

13

"

1 5

"

17

"

19

.

'

2 1

A

Number

FIGURE 8 Change in C H alpha shifts from ET-1 for ETM7A and ETD8A.

In addition, smaller temperature coefficients were measured for the amide protons of ET-1 in aqueous acetonitrile and 10% aqueous acetic acid solutions supporting the conclusion that these protons may be involved in hydrogen bonding (41, 43). Most of these protons were still present in both ET-1 and ETM7A after 45 min, as Figs. 5 and 6 show. In contrast, as Fig. 7 demonstrates, (some of) these protons in ETD8A were completely exchanged at the end of 45 min. This data suggests that the helical region in ETD8A is more accessible to solvent and is not as strongly hydrogen bonded when compared to ETM7A and the parent molecule.

0.4

-

0.2

-

E a

0.0 -

-3

-0.2

a

'

a

-

-0.4'

4-61

-0.8

-1.0' 1

b "

3

"

5

"

7

"

9

Residue

"

11

'

"

13

a

15

"

17

"

19

Number

FIGURE 9 Change in amidc shifts from ET-I for ETM7A and ETD8A.

5 20

zn

" 21

I

I

I

I

8.8

8.4

E.0

7,6

PPM FIGURE 10 Expanded regions from the NOESY spectra depicting differences in NOE intensity for selected NH-NH NOES in (A) ET-I and (B) ETDSA.

Endothelin- 1 conformation illustrated graphically in Figs. 8 and 9 which show the deviation of the respective CHcr and NH shifts of ETM7A and ETD8A from ET-1. The shifts for each residue of ETM7A, ETD8A, and ET- 1 were subtracted from random coil values (53), and then the mutant shifts were subtracted from those of ET-1. These delta values were then plotted versus residue number. The estimated error in chemical shift is approximately & 0.02 ppm. The ETM7A curve for the alpha protons is smooth, not deviating more than 0.05 ppm, suggesting strongly that the mutation has not induced a conformational change relative to ET-1. In contrast, Figs. 8 and 9 demonstrate substantial differences in NH chemical shifts between ET-1 and ETD8A with smaller shift differences for the CHa resonances. The CHa shift changes are localized to residues 3-12. The

0

€d

e

a

b

4.6

N H chemical shift changes occur in the same portion of the molecule, i.e. residues 3-13. These data imply that the mutation of Asp-8 to Ala-8 has induced a conformational change in the ‘‘loop’’ portion of the molecule, which contains both the type 1 p-turn and the first turn of the helical portion of the molecule. In order to determine whether these chemical shift changes observed in the ETD8A mutant were reflected in the sequential NOEs and in the medium and longrange NOE patterns, the NOESY spectra for all three molecules were examined in more detail. The structural features described earlier exist in all three molecules. On a qualitative basis, relative sequential NOE intensities for ET-1 and ETM7A are essentially identical as are the appearance of medium-range NOEs. In the case of ETD8A sequential NOE intensities are slightly per-

4.4

4.2

4.0

3.8

3.6

3:4

3.2

PPM FIGURE 11 Expanded regions from the NOESY spectra depicting differences in selected NOES for (A) ET-1 and (B) ETDIA. Square boxes in panel B show NOES that are missing in ETDBA.

52 1

D.C. Dalgarno et al. turbed when compared to ET-1 and ETM7A. In particular, as Fig. 10 shows, in EDT8A the d” connectivities between Ser-5 and Leu-6 are more intense relative to those of ET-1, while those between Asp-8 and Lys-9 are less intense than those of ET-1. In addition, as Fig. 11 demonstrates, the drN connectivity between Ser-5 and Leu-6 in ETD8A is weaker relative to that of ET-1. Fig. 11 also shows that the mediumrange NOE between Cys-3 p-CH2 and Ser-5 NH in ET-1 are lost in ETD8A as is the d,N(i + 2) connectivity between Ser-5 and Met-7. These changes can be interpreted as being due to differences in the orientation of the type I 8-turn spanning residues Ser-5 to Asp-8 with respect to both the N-terminal portion of the molecule and the helix. It is interesting to note that of these two mutants, ETD8A, with the mutation at a conserved position in the ETjSFTX family, appears to be the most conformationally perturbed. This suggests the possibility that Asp-8 is a pivotal residue in that it serves as the N-terminal residue for the helical segment. In addition to sequence position, conformational changes may arise from differences in sidechain interactions. Changing Asp-8 to Ala-8 results in a change in sidechain polarity, whereas the Met-7 to Ala-7 mutation is substituting one nonpolar sidechain for another. The effect of polarity may be important. Previous work where Met-7 of ET-1 was changed to norleucine induced very little conformational change relative to ET-1 (41). We have compared the conformations of these endothelin molecules in solution using three different NMR parameters: chemical shift, N O E patterns, and hydrogen-deuterium exchange. Chemical shift data provides the clearest information about conformational differences; however, it is not possible to interpret these differences in simple structural terms. Our present studies indicate that on a chemical shift basis ET-1 and ETM7A are extremely similar, whereas ETD8A has a perturbed conformation in the region between residues 3 and 12. In the case of ETDBA qualitative changes in the pattern of the NOEs and in their intensity provides some indication that rearrangements in the orientation of the type 1 B-turn with respect to both the N-terminus of the molecule and the helix are responsible for the observed chemical shift changes. Describing this change in terms of precise structural differences is difficult for several reasons: a paucity of long range NOE data, whether induced by conformational averaging or otherwise, and the necessity to rely on subtle intensity changes in sequential NOEs to indicate structural differences. The exchange studies support conformational differences between ET-1 and ETD8A resulting in a destabilization of the helical conformation with respect to N H exchange. The structural changes observed with the Asp-8 to Ala-8 change indicate that this mutation induces widespread structural perturbations in a major portion of the molecule. Such data is consistent with the conserved nature of Asp-8 in the ETiSFTX family. 522

We are currently investigating the conformation of ET-1 itself in more detail, particularly in the “loop” region. We are particularly interested in determining whether particular residues exhibit different dynamic properties resulting in the observation of averaged conformational properties. We hope to report on these studies and the effect of mobility on describing the structural effects of mutations in a future publication. ACKNOWLEDGMENTS We would like to thank Dr. J. Prestegard and Dr. Yu-sen Wang for insightful discussions and comments. In addition, we would also like to thank Dr. P. Das and Dr. B. Pramanik for the mass spectral data. Dr. B. Neustadt’s commcnts and review of the manuscript and Dr. M. Czarniecki’s continued support of the endothelin project are also greatly appreciated.

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