determination of disulfide bridge pattern in ω-conopeptides

Inr. J . Prpfide Prorem Rrr. 46, 19Y5, 3211-325 Prinred in Belgium - ail nghrs re.Yen.ed

Cop>righr 0 Munhqprird l Y Y 5

INTERNATIONAL JOURNAL OF PEPTIDE & PROTtlN RESEARCH

ISSN 0367-8377

Determination of disulfide bridge pattern in co-conopeptides

DAVID CHUNG. SMITA GAUR. J O H N R. BELL. J . RAMACHANDRAN and LASZLO NADASDI

,Veures Corporatioil, Menlo Purk. Cal[foriiia. USA

Received 30 September 1994, accepted for publication 29 January 1995

Dedicated to the memory of Dr. Johannes Meienhofer

Synthetic versions of seven naturally occurring w-conopeptides were subjected to structural analyses in order to determine their disulfide bridge pattern. The method applied in this study uses a combination of amino- acid composition and peptide sequence analysis of various peptide fragments generated by different enzymatic digestions. A temperature modification in the Edman degradation cycles of a protein sequencer allowed the unambiguous detection of the cleavage of cystine residues. The appearance of the cystine residues in particular cycles of the sequence analysis was characteristic of one or several of the theoretically possible 15 isomers. In the case of multiple choices, possible isomers were further eliminated by the amino-acid and sequence analysis of peptide fragments generated by the enzymatic digestion. All synthetic peptides, SNX-111, -157, -159, -183, -185, -230 and -231, were found to have the same disulfide bridge pattern as determined for the naturally occurring o-conopeptide G-VI-A, i.e. disulfide bridges between the half-cystines 1-16, 8-20 and 15-25 (using the amino-acid numbering of S N X - I l l ) . 0 Munksgaard 1995.

Key words: conopeptide; disulfide bridge determination: enqnie digestion

w-Conopeptides are a group of basic peptides, rang- ing in size from 24 to 27 amino-acid residues, found in the venoms of several species of fish-hunting marine snails (1). The w-conopeptide G-VI-A isolated from the venom of Conus geographicus has been a valuable pharmacological tool in the identification and charac- terization of N-type voltage-sensitive calcium channels (VSCC) in the neuron system (2, 3). A synthetic peptide, SNX-111, corresponding to the sequence of the w-conopeptide M-VII-A isolated from the venom of Conus magus, has recently been reported to be a potent

Abbreviations: TFA, trifluoroacetic acid: ACN. acetonitrile: IPA. isopropyl alcohol; PTH-Cys, the phcnylthiohydantoin derivative of cystine. The abbreviations for amino acids are according to ICPAC- IUB recommendations. VSCC. voltage-sensitive calcium channel: RP-HPLC, reversed-phase high-pressure liquid chromatograph! : ChTr, chymotrypsin; Tr, trypsin: SP, subniaxillaris protease: TL. thermolysin. The annotations for the synthetic versions of the naturally occurring o-conopeptides are as follows: SNX-111. M-VII-A;

185, T-VI-A; SNX-230. M-VII-C: SNX-23 1. [H>p']-M-VII-C. The SNX numbers refer to the synthetic versions of the naturall! occurring conopeptides (such as M-VII-A, S-VI-A. etc.). Since the disulfide bridging of the conopeptides iwlated from conus vcnunis n as not determined, we prefer the use of SNX nomenclalure specif!ing the disulfide bridging of the synthetic versions.

320

SNX-157, S-VI-A; SNX-159, M-VII-B: SNX-183. S-VI-B: SNX-

neuroprotective agent in animal models of global is- chemia (4). Novel w-conopeptides from other Conus species have been isolated (5) or their structures deduced from the cDNA sequences of genes coding for 0)-conopeptide precursors (6).

An important common structural feature of this group of peptides is the presence of six half-cystine residues organized into three disulfide bridges. Since three disulfide bonds can be formed in 15 distinct arrangements (Fig. l), determination of the disulfide bridge pattern, especially in the case of synthetic w-conopeptides and their analogs, is essential for mean- ingful correlation of structure and function. To date, the only information available regarding the disulfide arrangement of w-conopeptides is for the synthetic peptide corresponding to the deduced sequence of G-VI-A. Its disulfide bridge pattern according both to a chemi- cal method (7) and to 2D-NMR spectroscopy investi- gation (8) corresponds to that of isomer 8 in Fig. 1. In all work with w-conopeptides this structure has been implicitly assumed to be the characteristic one for various other conopeptides, though this has not been rig- orously determined.

As part of a program aimed at developing highly selective antagonists of new classes of ncuronal VSCC which have recently been identified by molecular clon- ing strategies (summarized in rcfs. 9 and 10) and elu-

Disulfide bridges in a-conopeptides

I

u 4

7

I 10

13

m

2

4 5

3

u 6

I FIGURE 1 The 15 possible isomers of a peptide coniaining three disulfide bridges. The folding pattern of the synthetic versions of the naturally occurring wconopeptides investigated in this papcr corresponded to isomer 8.

cidating the structural features of SNX-111 responsible for its selective interactions with ,%type VSCC, we here report the determination of the disulfide arrangement of several synthetic wconopeptides (1 1). In order to elu- cidate the disulfide bond organization of these peptides, they were submitted to digestion with one or the combination of some of the following enzymes: chymotrypsin, trypsin, submaxillaris protease and thermolysin. The peptide fragments were \eparated by reversed- phase HPLC, analyzed by amino-acid analysis and sequencing. In this strategy, the release of PTH-cystine at particular cycles of Edman sequencing is characteristic of the disulfide bridge pattern, thus providing the assignment of disulfide links. Similar sequencing tech- niques have been applied in detcrmining disulfide patterns of isolated peptides/proteins in combination with either enzyme digestions (12, 13) or with partial disulfide bond reductions, at low pH, followed by alkylation of the free sulfhydryls (14). It should be noted, that in the case of multiple disulfide bi-idge-containing peptides, several theoretical folding patterns may yield two or more isolated peptide fragments upon enzyme digestions, and a simple amino-acid analysis of these fragments may be enough to eliminate or include cer- tain isomers. The strategy we ;ire describing in this paper allowed the unambiguous determination of the disulfide arrangements in a number of structurally di- verse synthetic m-conopeptides.

EXPERIMENTAL PROCEDURES

RP-HPLC analysis and separations were carried out on a system consisting of two model llOB pumps, a model 421 controller and a model 165 variable- wavelength detector (Beckman, Palo Alto, CA)

equipped with a 4.5 x 250 mm Vydac 218 TP104 col- umn (C18,300 Apore size, 10 pmparticle size). Amino- acid analysis was performed on a model 119C amino- acid analyzer (Beckman, Palo Alto, CA).

Peptide sequencings were accomplished on a model 477A protein sequencer (ABI, Foster City, CA). The sequencing protocol on this instrument was modified by reducing the conversion flask temperature (from 64 to 50 "C) in order to minimize the destruction of the PTH-Cys. The conversion flask drying times were ad- justed appropriately to effect complete drying at the lower temperature.

The peptides investigated in this paper were synthesized by solid-phase peptide synthesis procedure described elsewhere (4, l l ) and obtained in 5- 15 % over- all yield as the major product following random air oxidation of the peptide after cleavage from the resin support and removal of all protecting groups by liquid HF. Amino-acid analysis indicated that the products had the expected composition.

Enzyme digestions of SNX-111 and analysis of its fragments. SNX-111 (100 pg, ca. 30 nmol) was dissolved on 100 pL phosphate buffer (pH 7.0, 10 mM). Chymot- rypsin (4 pg) was added and the solution was kept at 37 "C for 6 h. The digested peptide was then subjected to RP-HPLC analysis, and two major peaks were isolated (Fig. 2).

Amino-acid analysis of the two major components indicated that the peptides were, respectively: des- Met12Tyr13-SNX-l 11 (ca. 10 nmol) and the dipeptide Met-Tyr. For sequencing purposes the samples were desalted by RP-HPLC with the following solvents: A, 0.1 % TFA in water; B, isopropanol. This second RP-HPLC also served as an additional purification of the peptide. The sequencing data for d e ~ - M e t ' ~ T y r I ~ -

32 1

D. Chung et al.

RBSORBRNCE SOLVENT B

0.50

0.40

0.30

0.28

0 . 1 0

0.00

II rn r.

50%

4 0 %

30%

20%

10%

4 0 12 16

T I M E Cminl

FIGURE 2 RP-HPLC of the chymotryptic digest o f SNX-111. Solvent A: 5 mM potassium phosphate (pH 2.74); Solvent B: acetonitrile. The solid line is the recording at 210 nm, the dashed line is the recording at 280 nm normalized by the maximum signals to the chromatogram at 214 nm. The dotted line indicates the gradient formed with solvent B. The peak eluting at 7.9 min corrcsponds to d e ~ - M e t ] ' T y r ~ ~ - S N X - l l l (note the low absorbance value at 280 nm), while the peak eluting at 15.1 min is the dipeptide Met-Tyr.

SNX- 11 1, shown in Fig. 3, indicate that disulfide-linked cystines were cleaved off in cycles 3, 8 and 12.

Submaxillaris protease digest of des-Metl2Tyrl3- SNX-I 11 was performed as follows. The peptide(50 pg, ca. 17 nmol) was dissolved in 50 pL phosphate buffer (pH 7.0,10 mM). The enzyme (5 pg) was added and the

solution was incubated at 37 "C for 4 h. RP-HPLC of the digest is shown in Fig. 4. The desired, cleaved des- Met1*TyrI3-SNX-l 11 eluted at 8.5 min. This component was lyophilized and rerun in the isopropanol/TFA system for sequencing: the disulfide-linked cystines were cleaved off in cycles 3, 4 and 8 (Fig. 5).

300 t

v

a I t-

100 !

t - 5 10 15

EDMRN DEGRRDRTION CYCLES

FIGURE 3 The sequencing of des-Met"Tyr13-SNX-l 1 I : the amount of PTH-Cys in cycles 3 . 8 and 12 incrcased significantly, indicating cithcr the simultaneous cleavage or the cleavage of the second residue o f qst ine.

3 22

Disulfide bridges in o-conopeptides

TABLE 1 Summary of the principles applied in the disul@e bridge determinations of the oconopeptides discussed in this paper. Vertical lines indicate the

enzyme cleavage sites

Compounds and enzyme cleavage sites Enzyme Edman cycles Alternative isomers" cleaving PTH-cys

SNX-111: C-K-G-K-G-A-K-C-S-R-I~-M-Y-D-C-C-T-G-S-C-R-S-G-K-C I 1

I 1 1 I I I I I I

SNX-157: C-R-S-S-G-S-X-C-G-V-T-S-I-C-C-G-R-C-Y-R-G-K-C-Td I I 1 I 1 I

SNX-159: C-K-G-K-G-A-S-C-H-R~r-S-Y-D-C-C-T-G-S-C-N-R-G-K-C 1 1 I I I I

1 1 I 1 1 SNX-183: C-K-L-K-G-Q-S-C-R-K-I-S-Y-D-C-C-S-G-S-C-G-R-S-G-K-C

I I / I / 1 1 I I I

SNX-185: C-L-S-X-G-S-S-C-S-X-T-S-Y-N-C-C-R-S-C-N-X-Y-S-R-K-C-Rd

I I I I 1 SNX-230: C-K-G-K-G-A-P-C-R-K-T-M-Y-D-C-C-S-G-S-C-G-R-R-G-K-Ce

I t I l l I 1

! I I ! 1 1 !

ChTr ChTr, SP Tr

SP, TL

ChTr ChTr, SP Tr

ChTr ChTr, Tr

ChTr ChTr, Tr

ChTr ChTr, SP Tr

3,8, 12 3,4, 8 b

3c

3,8, 12 3, 8 b

3,1, 13 2, 3.7

3 , 4 , 8 2,3, 8 d

3,8, 13 3 , 8 b

1,7, 15 15

None

1,7, 15 15

9, 12, 15 5

15 1, 2,3, 5-7,9, 10, 12

1,7. 15 15

a The appearance of the PTH-Cys in the particular cycles was in agreement in each case with the folding pattern represented by isomer 8. Was not sequenced: the amino-acid analysis was in agreement with the fragment deducted from isomer 8 indicating the presence of six half-cystine residues in the major digested component, thereby isomer 15 can be eliminated (see text for further dctails). Another fragment, SSGSXCG ... C was isolated but not sequenced because by composition it corresponded to segment ( 3 - 9 + 18). The amino acid composition of the digested and isolated product corresponded to that of isomer 8, the other alternative isomers whould not have yielded one peptide containing three cystines. X, hydroxyproline. SNX-231, a variant of SNX-230 contining Hyp in position 7, was subjected to the same analyses resulting in the same folding pattern.

Disuljide bridge determinations of SNX-157, -159, -183, -185, and -230. The peptides were digested by the various enzymes, and the fragments were isolated from the digest by RP-HPLC procedures as described above. The strategies and the principles for these peptides are summarized in detail in Table 1.

RESULTS AND DISCUSSION

Random oxidation of a conopeptide containing six cys- teine residues can result in the formation of one or more of the fifteen possible disulfide isomers illustrated in Fig. 1. However, when the synthetic conopeptides, studied in this paper, were subjected to random air oxidation after cleavage from the solid support and removal of all protecting groups by liquid H F we obtained only one major product. Based on the amino- acid sequence of the peptide, a suitable series of enzymatic treatments was adopted to generate fragments which could be identified by amino-acid analysis after separation on reversed-phase HPLC. Sequencing analy-

ses of these peptide fragments led to the release of PTH-Cys at a unique and predictable set of cycles for each of the isomers. This method utilizes the fact that as long as disulfide bonds remain intact, when the first half of a cystine residue is cleaved from the peptide during the Edman degradation, it remains attached to the remaining peptide chain through the disulfide bond to the second half. When the sequential cleavage of the sequencing reactions reach the second half cystine, the cystine residue is released from the peptide chain and can be detected (12, 13). Of course, in the case of a disulfide-linked peptide with two free N-termini (as a result of an enzyme digestion, for example), it is possible for both half cystines to be cleaved at the same time. The analysis depends upon predicting when the cystine pairs will be released from the various possible disulfide-bonded arrangements upon Edman degradation and ruling out disulfide patterns which are incompatible with the experimental results. Using the HPLC protocol recommended by the manufacturer the PTH- Cys coelutes with the PTH-Tyr, but in case of each of

323

D. Chung et nl.

-

.

-

-

I

RBSORBRNCE

50%

40%

..30%

20%

10%

m m

SOLVENT B

4 8 12 16

T I M E C r n i n l

FIGURE 4 RP-HPLC of the submaxillaris protease digest of des-Met12Tqr13-SNX-l11. The chromatographic conditions are identical to those in Fig. 2. The peak eluting at 8.9 min corresponds 10 des-Met12T>r'3-SNX-l 1 1 cleaved at several sites by the enzyme (Table 1).

the peptides discussed in this paper this interference was resolved by the removal of the tyrosine residue(s) during the enzyme digestions. When this was not possible, we found that a convenient modification of the HPLC protocol resulted in a sufficient separation between these two amino-acid derivatives. In the case of the intact peptides, the predicted appearance of the PTH-Cys derivatives would not identify unambiguously

the individual isomers; moreover, the PTH-Cys derivatives would not be easily determined after 20 cycles owing to the low yield of the recovery. Therefore a series of enzyme digestions was performed on each intact (disulfide-bridged) peptide; the analyses of the resulting various fragments defined the disulfide bridge pattern in the original peptide unambiguously (Table 1). The strategy is illustrated below for SNX-111.

5 ! 0 15

EDMAN DEGRRDRTION CYCLES

FIGURE 5 The sequencing of d e ~ - M e t ~ ~ T y r l ~ - S N X - l l l cleaved at positions 10 and 11: the amount of PTH-Cys in cycles 3, 4 and 8 increased significantly, indicating either the simultaneous cleavage or the cleavage of the second residue of cystine.

3 24

Disulfide bridges in w-conopeptides

encoded by the particular positions of the half-cystine residues in the linear sequence: from the point of view of the natural folding pattern, this arrangement makes the structures highly tolerant toward even drastic structural changes.

Disulfide bridges in SNX-I I 1 SNX-111 was treated with chyniotrypsin as described under Experimental Procedures and the digest con- tained two major components, which according to the amino-acid analysis corresponded to the dipeptide Met- Tyr and des-Metl2TyrI3-SNX-1 11. The generation of des-Metl2Tyrl3-SNX-l 11 containing all three disulfide bridges by chymotryptic cleavage of SNX-111 eliminates three possible arrangements, namely, isomers 1-3, shown in Fig. 1 (these peptides would fall apart). Sequence analysis ~ f d e s - M e t ' ~ T j r ~ ~ - S N X - l 11 showed that PTH-Cys was released at cycles 3,8 and 12 (Fig. 3). This result eliminates all the isomeric arrangements, except 7, 8 and 15. Digestion of SNX-111 by chymotrypsin and trypsin yielded one component with all the cystines; this result is incompatible with the disulfide arrangement in isomer 15. To resolve whether isomer 7 or 8 represented the disulfide arrangement in SNX- 11 1, des-Meti2Tyrl3-SNX-1 11 was further digested with submaxillaris protease, which cleaves at the car- boxyl side of arginine residues (Fig. 4). Sequence analysis of the product isolated from this digest revealed PTH-Cys in cycles 3, 4 and 8 (Fig. 5), confirming that SNX-111 has the disulfide bridging of isomer 8. Isomer 7 would have released PTH-Cys at cycles 3, 7 and 8.

Disulfide bridges in other o-conopcJptides Similar strategies were used to establish the pairing of the cystines in the synthetic versions of other w-conopeptides found in the venoms of several Conus species (Table 1). The disulfide arrangements of SNX-

B), SNX-185 (T-VI-A) and SNX-230 (M-VII-C) were all identical to that of SNX- 11 1. namely, isomer 8 in Fig. 1. The strategy used is effective, especially when half cystines are next to each other.

The peptides investigated in this work represent a large amino-acid variability in two thirds of the sequence positions. Yet the highlj conserved locations and spacing of the cystine residues, as well as some other conserved amino acids, should provide a unique three-dimensional frame for the specific disulfide bridge pattern characteristic for the w-conopeptides. We have synthesized a large number of analogs of the above naturally occurring w-conopeptidcs (even the conserved non-half cystine residues were displaced) in order to define the functional roles of the various amino-acid side chains. This approach obviously depends on the analog being as similar as possible in structure to the reference molecule, differing only in the replacement side chain. In this regard, it is perhaps remarkable that (as we have shown in this report) the majority of the analogs we have designed have preferentially folded into a conformation which, based upon the disulfide arrangement, is identical to the native conopeptide. This finding underlines the significance of the information

157 (S-VI-A), SNX-159 (M-VII-B), SNX-183 (S-VI-

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1453- 1463

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325

determination of disulfide bridge pattern in ω-conopeptides

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