The Structure of Ro 09-0198 in Different Environments H. KESSLER,' D. F. MIERKE,'
J.SAULITIS,' S. SEIP,' S. STEUERNACEL,' T. WEIN,' and M. W I L L 3
'Organisch Chemisches lnstitut der Technischen Universitat Munchen, Lichtenbergstrasse4, D-8046 Garching; *Bruker Analytische Messtechnik G m b H , Silberstreifen, D-7512 Rheinstetten 4; and 3BASF AG, D-6700 Ludwigshafen, Germany
SYNOPSIS
The constitution and configuration of Ro 09-0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09-0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.
INTRODUCTION
H20
Ro 09-0198 (cinnamycin) is a quadricyclic nonadecapeptide that contains several uncommon amino acids, such as lanthionine, methyllanthionine, lysinoalanine, and p( OH) aspartic acid. The separation of hydrophilic and hydrophobic residues results in an amphiphilic character. Investigations by others'Z2 have shown that cinnamycin interacts specifically with some membrane compartments. Here, nmr studies in different environments (H20, SDS micelles/H20, SDS bilayer) have been done to elucidate the conformational changes that might occur when the surrounding of the peptide is changed.
NMR IN H 2 0 AND BOUND TO SDS MICELLES Resonance assignments were made using homonuclear [correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY)3 and heteronuclear [ heteronuclear multiple quantum correlation (HMQC), HMQC-TOCSY ] techniques. Complete assignment of protons and carbons was achieved for all isotropic solvents (DMSO, H20, SDS/H20). ~
Biopolymers, Vol. 32, 427-433 (1992) 0 1992 John Wiley & Sons,Inc
CCC 0006-3525/92/040427 07$04.00
JNH.Ha and 3JH,.Hs coupling constants were obtained from exclusive COSY and double quantum filtered COSY spectra. Distances were derived from a single NOESY spectrum (100 ms mixing time). Diastereotopic assignment for 7 of 10 nonisochronous /3-methylene proton pairs (&protons of Gln 3 , AsnI7,and N'-Ala6 are isochronous) have been made on the basis of homonuclear coupling constants and NOE effects. 3
SDS/H2O The large line width in SDS/H20 prevents the accurate determination of coupling constants from homonuclear spectra. Therefore only NOE data were used for determination of the conformation bound to the micelle. Strong spin diffusion required the measurement of NOE buildup. Distances were obtained by the following: 1 Fitting cross-peak intensities of a series of
two-dimensional NOESY spectra, taken at different mixing times to a quadratic equation, in close analogy to a method proposed by Wagner et al.3 2. Integration of cross-peaks from a three-dimensional (3D) NOE spectrum and correction of spin diffusion, utilizing 3D crosspeaks.4 427
428
KESSLER E T AL.
Table I Distances of NOESY Spectra and MD Calculations in Vacuo
SDS Proton I Ala(S)'-H" Ala(S)'-Ha Ala(S)'-Ha Ala(S)'-H" Ala(S)'-Ha Ala(S)'-H" Ala(S )'-Ha Ala(S)'-HBrS Ala(S)'-HB,R Ala(S)'-HBSR Ala (S) '- HB*' Ala(S)'-HBvS Ala(S)'-HBsS Ala(S)'-HBsR Arg-NH A&-NH Arg-NH Arg-NH Arg-NH Arg-NH A&-NH A$-NH Arg-NH Arg-NH Arg-NH Arg-NH A$-Ha Arg-Ha A&-H" A&-Ha Arg-H" Arg-Ha Arg-HBSS Arg-HBrR Arg-HB,S Ar2-HBsR Gln3-NH Gln3-NH Gln3-NH Gln3-NH Gln3-NH Gln3-NHa Gln3-NH" Gln3-NH" Gln3-NH" Gln3-NH" Gln3-NH" Gln3-NHB Gln3-NHB Gln3-NHB Gln3-NHB Gln3-NHB Gln3-NH7
Proton 2 Ala(S)'-HBS Ala(S)'-HP,R Arg-NH Lyslg-NH Lys'g-HB,S Lys"-H@*R LysIg-Hy,S Arg2-NH Arg-NH Abu(S)18-NH Abu(S)"-NH Abu(S)l8-Hy Lydg-NH Lyslg-NH Arg-Ha Ar$-HB,S Arg-HBsR Gln3-Ha Gln3-HB AS~'~-H~ Abu(S)18-H4 Lyslg-NH L~S'~-H" Lys'9-HB.S Lyslg-HO*R Lyslg-Hy,S Arg-HB*S Ar2-HBsR Arg-H6sS Gln3-NH Gln3-HB Gln3-H7 Gln3-NH Gln3-NH Gln3-Ha Gln3-H" Gln3-H" Gln3-HB Gln3-H7 Asn17-H" L~S'~-H~,S Gln3-H7 Ala(S)4-NH G1y"-NH Asn17-NH Asn17-H" Abu(S)"-NH Ala(S)4-NH Ala(S)4-HB.S As~'~-H" Asn17-HB Abu(S)18-NH Ala(S)4-NH
H20
NOE
MD
-
_.
-
-
240-270 290-460 200-460 200-460 200-500 270-400 270-400 410-510 305-465 200-360 200-360 290-310 302-332 243-273
216 340 414 268 426 415 386 450
-
-
292-322 -
250-280 278-308 400-450 230-260 400-500 400-500 247-277 294-324 379-439 358-418 229-259 250-350 280-380 400-460 200-350 190-220 350-460 226-256 350-390 263-363 -
300-430 338-518 304-404
-
411 444 340 287 363 250 -
313 257 301 425 226 498 515 276 389 435 483 291 300 391 477 -
292 234 481 225 359 348 447 530 -
454
NOE
MD
279-319 307-337 272-302
262 296 232 462 257 383 276 318 312 438 289 358 295 428 582 614 555 246 482 430 258 323 261 285
-
351-419 212-252 295-335 396-446 230-350 272-312 378-428 272-302 308-358 216-256 370-410 476-596 401-511 482-522 285-325 403-443 435-475 299-339 390-430 275-315 215-255 375-415
-
238
-
-
-
-
-
285-318 245-355 -
288 295 -
-
-
449-489 306-416 201-241 408-448
560 307 211 504 227 390 463 543 -
-
285-325 277-387 351-461 368-478 -
STRUCTURE OF RO 09-0198
429
Table I (Continued) SDS Proton I Gln3-NH7 Gln3-NHy Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)' -NH Ala(S)'-NH Ala(S)4-H" Ala(S)'-H" Ala(S)4-H" Ala(S)'-H" Ala(S)'-HB.' Ala(S)'-HBVR Ala(S)'-HBSS Ala(S)4-HB*S Ala(S)'-HBoS Ala(S)'-HB*' Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-HB*' Ala(S)'-HB*' AIa(S)5-HB*S Ala(S)6-HB3R Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-HB Phe7-NH Phe7-NH Phe7-NH Phe7-NH Phe7-H" Phe7-H" Phe7-H"'
Proton 2 Asn17-H" AS~'~-H~ Ala(S)'-H" Ala(S)4-HB*S Ala(S)'-HBsR Ala(S)"-H" Ala(S)"-HBsR Asn17-NH Ala(S)4-HB*S Ala(S)4-HB~R Ala(S)'-NH Abu(S)"-HY Ala(S)'-NH Ala(S)5-NH Ala(S)I4-NH Ala(S)14-H" Ala(S)14-HBsR Asp"-NH Ala(S)'-H" Ala(S)'-HB8' Ala(S)'-HOsR Ala(S)14-H" Ala(S)14-HB.S Ala(S)14-HBvR Ala(S)'-H09' Ala(S)5-H0*R Ala6-NH Ala6-H" Ala6-HB Abu(S)ll-HB Ala(S)14-H" Ala6- N H Ala(S)14-NH Abu(S)"-H@ Abu(S)"-HP Ala6-H" Ala6-HB Phe7-NH Gly8-N H Asp"-NH AsP'~-O*' A~p'~-0'* Lys'g-Hy.S Lys'g-H'*' Lys'9-H'~~ Phe7-NH Phe7-H" Phe7-HB*' Phe7-HB*R G ~ ~ - N H Phe7-H0ss Phe7-H0tR G~~-NH
H*0
NOE
MD
NOE
MD
300-430 357-537 290-310 350-450
370 427 289 329
303-343 360-400 -
-
-
361-391 396-446 380-460 232-262 240-270 288-318 290-400 287-317 -
287-307 302-332 -
-
418 506 361 247 216 220 54 1 397 285 353 -
-
-
259-289 270-300 268-298 350-460 327-447 200-269 350-540
248 301 213 424 496 347 428
-
-
-
-
214-244 256-286 290-310 288-388 231-261 307-337
265 395 29 1 296 211 355
-
-
170-220 170-220 -
-
286 303 -
-
-
262-362 297-317 290-320 272-302 299-319
282 256 280 219 327
-
-
273-303
-
249
235-275 283-323 238-278 326-366 256-296 329-369 253-293 366-406 467-507 -
-
279 290 258 297 229 413 356 548 379 435 453
-
m-rn
9457.
222-262 296-336 378-408 380-420 235-265 258-288 222-262 -
222 274 480 465 299 246 212 -
335-475 435-475 280-395 282-322 -
316-356 170-220 170-220 345-495 345-495 345-495 316-356 385-425 260-300 255-295 305-345
431 547 274 268 318 285 222 599 453 504 317 368 233 251 417
430
KESSLER ET AL.
Table 1 (Continued)
SDS Proton I Phe7-HBSR Phe7-H"' Phe7-HBSR GlyS-NH Gly8-NH Giy8G ~ ~ ~ - H ~ ~ 1 ~ ~ G ~ ~ ~ - H ~ . ~ Prog-H" Prog-H" Prog-H* P ~ ~ ~ - H ~ , ~ POrg-H"' P~o~-H~,~ pro9-H6*s Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-H" Phe'O-H" Phe'O-H" Phelo-HA*S p he'o- H@sR Phe'o-H"' Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu(S)''-H" Abu(S) -Ha Abu(,)"-He Abu(S)"-HB Abu(S)" -HY Phe'*-NH Phe'*-NH Phe'2-NH Phe'I-NH Phe'*-NH Phe'2-H"' Phe'2-H"R ValL3-NH Val13-NH Val"-NH ValI3-NH ValL3-H" Val13-H" ValI3-H" Val13-H" Va1I3-H"
Proton 2
~
~
HzO
NOE
MD
NOE
MD
290-320 294-384 310-400 202-252 219-269 -
374 475 338 292 236 244 297 354 419 311 284 293 225 250 45 1 728 441 -
342-382
353 277 279 322 290 226 291 -
194-234 262-292 321-351 350-460 350-460 -
280-310 237-267 263-293 290-320 255-365 400-600 400-600 317-357 251-281 289-329 350-460 298-328 215-275 210-240 250-360 330-370 306-336 266-356 272-292 242-272 213-243 205-235 323-423 266-296 270-300 267-287 215-245 321-421 224-324 235-255 228-338 200-310 200-230 -
342 311 346 485 212 288
-
246 379 332 415 344 290 306 248 226 402 294 319 275 242 431 277 23 1 304 379 212 -
-
271-311 290-310 291-331 246-286 278-318 231-271 272-312 398-438 402-442 323-443 355-475 275-365 275-365
-
303 453 400 477 516 372 332 -
-
-
-
252 281 302 363 279 363 330 327 226 293 -
275-315 275-315 244-314 289-329 229-269
-
345-385 235-355 245-285 224-264 242-362 233-273 338-378 335-353 285-310 250-290 285-325 275-415 275-415 195-245 375-445
246 400 315 284 303 260 343 311 235 447
STRUCTURE OF Ro 09-0198
Table I
431
(Continued) SDS
Proton I Val13-H" Val13-H" Val13-HB va113-~73
Val'3-HY,R Ala(S)14-NH Ala(S)14-NH Ala(S)I4-NH Ala(S)14-NH Ala(S)14-Ha Ala(S)14-HB3s Ala(S)14-HB*R A~P-NH Asp15-NH Asp"-NH Asp15-NH Asp"-NH Asp"-H" G ~ ~ - N H Gly"-NH G~~~G-H".S
G~~~G-H*,R
G~~~G-H",R Asn17-NH AsnI7-NH Asn17-H" Asn17-H" Asn17-HB Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu ( S )''-Ha Abu(S)"-H" Abu ( S )''-Ha Abu(S)"-H4 Lyslg-NH Lyslg-NH Lyslg-NH Lyslg-NH Lyslg-NH LysIg-NH Lyslg-H" Lyslg-H" Lyslg-H" Lyslg-H"
Proton 2 Ala(S)14-HPsS Ala(S)14-HB9R Ala(S)14-NH Ala(S)14-NH Ala(S)14-NH Ala(S)14-Ha Ala(S)14-HB,S Ala(S)'4-H@3R Asp"-NH Asp"-NH Asp"-NH Asp15-NH Asp15-H" Asp15-H@ GlylG-NH G~~~G-H".s GlylG-H".R G1y1'-NH G~~~G-H".S Asn17-NH Asn17-NH Lyslg-Hy-S Asn17-H" As~'~-H@ Abu(S)"-NH Lyslg-NH Abu(S)"-NH Abu(S)l'-H" Abu(S)"-H@ Abu(S)18-Hy Lyslg-NH Lys'9-HO.R Abu(S)"-HB Abu(S)18-Hy Lyslg-NH Lyslg-NH Lyslg-H" Lyslg-H@,S Lys'g-H@,R Lys'g-Hy,S Lyslg_Hy,R Lys'9-H Lys'g-H@,s
Lys'g-H@,R Lys'g-Hy,S Lys"-H6
H20
NOE
MD
NOE
MD
-
-
250-350 268-398 269-289 240-290 250-300 222-252 316-366 346-392 290-310 342-372 250-280
352 497 288 304 256
442-472 372-412 452-492 -
530 401 43 1 -
-
335-365 280-310 285-305 301-321 -
270-290 220-340 208-248 250-380 300-460 270-310 337-367 266-376 234-264 395-425 230-255 231-291 -
258-278 270-300 -
215-245 233-263 400-500 219-249 234-264 270-300 380-480
-
208 373 426 284 394 252
-
323 262
-
340 224 291 306 221 392 392 285 354 356 244 459 245 278 274 347 219 321 560 256 304 313 455
-
-
272-312 210-250 231-271 348-388 204-254 380-420 395-435 285-310 321-361 355-405 395-465 415-445 255-295 245-285 415-455 293-313 241-351 219-259 279-389 339-379 258-378 225-265 263-303 285-405 314-354 328-368 273-319 295-335 226-266 326-336 -
270 329 195 43 1 195 374 448 285 385
-
446 477
-
-
525 321 216 462 292 316 242 334
-
355 291 269 200 303 350 398 279 355 234 278 -
-
-
231-271 294-334 -
259 302 -
-
432
KESSLER E T AL.
These distances, which are listed in Table I, were used in model building and restrained molecular dynamics (MD) for the structure determination.
MD CALCULATIONS All calculations were done with programs of the GROMOS ( GROingen Molecular Simulation) library5 on Silicon Graphics 4D/240SX and 4D/ 70GTB computers. A simulated annealing approach together with energy minimization ( E M ) was used to achieve preliminary structures from the given NOE data sets. The starting structure was taken from previous calculations of the structure in DMSO
s ~ l u t i o nFor . ~ ~both ~ calculations (H20 and SDS/ H20)we used the following scheme: -10 ps of high temperature MD at 1000 K with a force constant for the distance restraining potential of K h = 4000 kJ /mol- nm2. -10 ps MD for cooling down to 300 K and following EM. The preliminary structure in SDS /H20that results from MD calculations with 152 distance constraints (49 intraresidual, 38 sequential, 65 long range) differs in various aspects from the structure in H 2 0 (138 distance restraints, 41 intraresidual, 31 sequential, 66 long range). A comparison of the back-
Figure 1. Backbone atoms and sulfur bridges of cinnamycin. The structure on the left was obtained from NOEs measured in SDS/H20, the structure on the right from NOEs measured in HzO. The N- and C-terminal ends are oriented to the bottom. Nitrogens are stippled, oxygens and sulfur atoms are solid. The carboxyl group at the C-terminus is omitted for clarity.
STRUCTURE OF Ro 09-0198
433
STRUCTURE IN H20 AND SDS MICELLES
A Phe Harom /CHQ SDS
11
2
1
Phe Haom/ (CH2In SDS
Phe Harem/ HP
J
Phe Harem /(CHz-O-) SDS
I;
An inspection of the NOE table indicates that there is a change in the conformation when the environment is changed. A comparison of the backbone atoms of the result of restrained MD in vacuo using the experimental restraints as obtained in H20 and SDS micelles shows that the main difference is in a hinge region, which connects the hydrophilic part with the lipophilic one. The general structure of the @-sheetof the A ring (amino acids 1-4 and 14-19) is retained but the hydrogen-bond pattern has changed. It should be noted that the results have to be refined by MD calculations including the solvent. Our preliminary results indicate that in spite of the polycyclic skeleton there is still a lot of flexibility in the structure.
ORIENTATION IN MEMBRANES
Phe Harem /Ha
(NOE,exchange)
NH2’Hz0
l5
Gln3,Asn17 NH2 (NOE,exchange)
The amphiphilic nature of cinnamycin immediately led to the speculation of its orientation in a biological membrane. For a proof of this we have recorded proton-MAS spectra of cinnamycin in SDS bilayers. A NOESY spectrum under these conditions contains a set of highly resolved cross-peaks (Figure 2 ) between the aromatic protons of cinnamycin and the CH, groups of SDS lipophilic chains. This provides evidence that the lipophilic end of the peptide sticks in the lipophilic part of the bilayer. Financial support by the Deutsche Forschungsgemeinschaft and the Fonds der chemischen Industrie is gratefully acknowledged.
REFERENCES
ppm
7
6
Figure 2. Part of the 500-MHz ‘H MAS NOESY spectrum of cinnamycin in SDS bilayers. In F2 only the region of the NH and aromatic protons is shown. Measurement conditions: SDS-DIZ (25 wt% ) , SDS-HI, (6.5 wt% ) , cinnamycin (13.6 wt% ), H 2 0 (54.4 wt%) at 297 K, molar ratio SDS/cinnamycin = 18; mixing time = 100 ms. A jump return (JR) observing pulse in the NOESY sequence and 5-kHz MAS were used.
bone atoms is shown in Figure 1. Calculations in the corresponding environments are in progress and should lead to more refined structures.
1. Wakamiya, T., Fukase, K., Naruse, N., Konishi, M. & Shiba, T. (1988) Tetruhed. Lett. 29,4771-4772. 2. Wiltschek, R. (1991) Diplomarbeit, Prof. E. Sackmann, Technische Universitat Munchen. 3. Hyberts, S. G. & Wagner, G. (1989) J. Mugn. Reson. 81,418-422. 4. Kessler, H., Seip, S. & Saulitis, J. (1991) J. Biomol. N M R 1,83-92. 5. van Gunsteren, W. F. & Berendsen, H. J. C. (1987) Groningen Molecular Simulation (GROMOS), Library Manual, Biomos, Groningen. 6. Will, M. (1989) Ph.D. thesis, Frankfurt/Main. 7. Kessler, H., Steuernagel, S., Will, M., Jung, G., Gillessen, D. & Kamiyama, T. (1988) Helv. Chim. Actu 71,1924-1929.
Received July 1 , 1991 Accepted August 13, 1991
J.SAULITIS,' S. SEIP,' S. STEUERNACEL,' T. WEIN,' and M. W I L L 3
'Organisch Chemisches lnstitut der Technischen Universitat Munchen, Lichtenbergstrasse4, D-8046 Garching; *Bruker Analytische Messtechnik G m b H , Silberstreifen, D-7512 Rheinstetten 4; and 3BASF AG, D-6700 Ludwigshafen, Germany
SYNOPSIS
The constitution and configuration of Ro 09-0198 (cinnamycin) have been determined in DMSO. Further investigations in aqueous solution, in SDS micelles and in a lipid bilayer have been done to study the influence of different environments on the conformation of the peptide. It turned out that in spite of the polycyclic structure of the molecule, the conformation is drastically changed going from water to SDS micelles. Ro 09-0198 orients itself in lipid bilayers as expected from its amphiphilic structure. According to a nuclear Overhauser effect spectroscopy experiment under magic angle spinning (MAS) conditions, the molecule is incorporated into the membrane with its hydrophobic part inside the bilayer.
INTRODUCTION
H20
Ro 09-0198 (cinnamycin) is a quadricyclic nonadecapeptide that contains several uncommon amino acids, such as lanthionine, methyllanthionine, lysinoalanine, and p( OH) aspartic acid. The separation of hydrophilic and hydrophobic residues results in an amphiphilic character. Investigations by others'Z2 have shown that cinnamycin interacts specifically with some membrane compartments. Here, nmr studies in different environments (H20, SDS micelles/H20, SDS bilayer) have been done to elucidate the conformational changes that might occur when the surrounding of the peptide is changed.
NMR IN H 2 0 AND BOUND TO SDS MICELLES Resonance assignments were made using homonuclear [correlated spectroscopy (COSY), total correlation spectroscopy (TOCSY), nuclear Overhauser effect spectroscopy (NOESY)3 and heteronuclear [ heteronuclear multiple quantum correlation (HMQC), HMQC-TOCSY ] techniques. Complete assignment of protons and carbons was achieved for all isotropic solvents (DMSO, H20, SDS/H20). ~
Biopolymers, Vol. 32, 427-433 (1992) 0 1992 John Wiley & Sons,Inc
CCC 0006-3525/92/040427 07$04.00
JNH.Ha and 3JH,.Hs coupling constants were obtained from exclusive COSY and double quantum filtered COSY spectra. Distances were derived from a single NOESY spectrum (100 ms mixing time). Diastereotopic assignment for 7 of 10 nonisochronous /3-methylene proton pairs (&protons of Gln 3 , AsnI7,and N'-Ala6 are isochronous) have been made on the basis of homonuclear coupling constants and NOE effects. 3
SDS/H2O The large line width in SDS/H20 prevents the accurate determination of coupling constants from homonuclear spectra. Therefore only NOE data were used for determination of the conformation bound to the micelle. Strong spin diffusion required the measurement of NOE buildup. Distances were obtained by the following: 1 Fitting cross-peak intensities of a series of
two-dimensional NOESY spectra, taken at different mixing times to a quadratic equation, in close analogy to a method proposed by Wagner et al.3 2. Integration of cross-peaks from a three-dimensional (3D) NOE spectrum and correction of spin diffusion, utilizing 3D crosspeaks.4 427
428
KESSLER E T AL.
Table I Distances of NOESY Spectra and MD Calculations in Vacuo
SDS Proton I Ala(S)'-H" Ala(S)'-Ha Ala(S)'-Ha Ala(S)'-H" Ala(S)'-Ha Ala(S)'-H" Ala(S )'-Ha Ala(S)'-HBrS Ala(S)'-HB,R Ala(S)'-HBSR Ala (S) '- HB*' Ala(S)'-HBvS Ala(S)'-HBsS Ala(S)'-HBsR Arg-NH A&-NH Arg-NH Arg-NH Arg-NH Arg-NH A&-NH A$-NH Arg-NH Arg-NH Arg-NH Arg-NH A$-Ha Arg-Ha A&-H" A&-Ha Arg-H" Arg-Ha Arg-HBSS Arg-HBrR Arg-HB,S Ar2-HBsR Gln3-NH Gln3-NH Gln3-NH Gln3-NH Gln3-NH Gln3-NHa Gln3-NH" Gln3-NH" Gln3-NH" Gln3-NH" Gln3-NH" Gln3-NHB Gln3-NHB Gln3-NHB Gln3-NHB Gln3-NHB Gln3-NH7
Proton 2 Ala(S)'-HBS Ala(S)'-HP,R Arg-NH Lyslg-NH Lys'g-HB,S Lys"-H@*R LysIg-Hy,S Arg2-NH Arg-NH Abu(S)18-NH Abu(S)"-NH Abu(S)l8-Hy Lydg-NH Lyslg-NH Arg-Ha Ar$-HB,S Arg-HBsR Gln3-Ha Gln3-HB AS~'~-H~ Abu(S)18-H4 Lyslg-NH L~S'~-H" Lys'9-HB.S Lyslg-HO*R Lyslg-Hy,S Arg-HB*S Ar2-HBsR Arg-H6sS Gln3-NH Gln3-HB Gln3-H7 Gln3-NH Gln3-NH Gln3-Ha Gln3-H" Gln3-H" Gln3-HB Gln3-H7 Asn17-H" L~S'~-H~,S Gln3-H7 Ala(S)4-NH G1y"-NH Asn17-NH Asn17-H" Abu(S)"-NH Ala(S)4-NH Ala(S)4-HB.S As~'~-H" Asn17-HB Abu(S)18-NH Ala(S)4-NH
H20
NOE
MD
-
_.
-
-
240-270 290-460 200-460 200-460 200-500 270-400 270-400 410-510 305-465 200-360 200-360 290-310 302-332 243-273
216 340 414 268 426 415 386 450
-
-
292-322 -
250-280 278-308 400-450 230-260 400-500 400-500 247-277 294-324 379-439 358-418 229-259 250-350 280-380 400-460 200-350 190-220 350-460 226-256 350-390 263-363 -
300-430 338-518 304-404
-
411 444 340 287 363 250 -
313 257 301 425 226 498 515 276 389 435 483 291 300 391 477 -
292 234 481 225 359 348 447 530 -
454
NOE
MD
279-319 307-337 272-302
262 296 232 462 257 383 276 318 312 438 289 358 295 428 582 614 555 246 482 430 258 323 261 285
-
351-419 212-252 295-335 396-446 230-350 272-312 378-428 272-302 308-358 216-256 370-410 476-596 401-511 482-522 285-325 403-443 435-475 299-339 390-430 275-315 215-255 375-415
-
238
-
-
-
-
-
285-318 245-355 -
288 295 -
-
-
449-489 306-416 201-241 408-448
560 307 211 504 227 390 463 543 -
-
285-325 277-387 351-461 368-478 -
STRUCTURE OF RO 09-0198
429
Table I (Continued) SDS Proton I Gln3-NH7 Gln3-NHy Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)' -NH Ala(S)'-NH Ala(S)4-H" Ala(S)'-H" Ala(S)4-H" Ala(S)'-H" Ala(S)'-HB.' Ala(S)'-HBVR Ala(S)'-HBSS Ala(S)4-HB*S Ala(S)'-HBoS Ala(S)'-HB*' Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-NH Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-H" Ala(S)'-HB*' Ala(S)'-HB*' AIa(S)5-HB*S Ala(S)6-HB3R Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-NH Ala6-HB Phe7-NH Phe7-NH Phe7-NH Phe7-NH Phe7-H" Phe7-H" Phe7-H"'
Proton 2 Asn17-H" AS~'~-H~ Ala(S)'-H" Ala(S)4-HB*S Ala(S)'-HBsR Ala(S)"-H" Ala(S)"-HBsR Asn17-NH Ala(S)4-HB*S Ala(S)4-HB~R Ala(S)'-NH Abu(S)"-HY Ala(S)'-NH Ala(S)5-NH Ala(S)I4-NH Ala(S)14-H" Ala(S)14-HBsR Asp"-NH Ala(S)'-H" Ala(S)'-HB8' Ala(S)'-HOsR Ala(S)14-H" Ala(S)14-HB.S Ala(S)14-HBvR Ala(S)'-H09' Ala(S)5-H0*R Ala6-NH Ala6-H" Ala6-HB Abu(S)ll-HB Ala(S)14-H" Ala6- N H Ala(S)14-NH Abu(S)"-H@ Abu(S)"-HP Ala6-H" Ala6-HB Phe7-NH Gly8-N H Asp"-NH AsP'~-O*' A~p'~-0'* Lys'g-Hy.S Lys'g-H'*' Lys'9-H'~~ Phe7-NH Phe7-H" Phe7-HB*' Phe7-HB*R G ~ ~ - N H Phe7-H0ss Phe7-H0tR G~~-NH
H*0
NOE
MD
NOE
MD
300-430 357-537 290-310 350-450
370 427 289 329
303-343 360-400 -
-
-
361-391 396-446 380-460 232-262 240-270 288-318 290-400 287-317 -
287-307 302-332 -
-
418 506 361 247 216 220 54 1 397 285 353 -
-
-
259-289 270-300 268-298 350-460 327-447 200-269 350-540
248 301 213 424 496 347 428
-
-
-
-
214-244 256-286 290-310 288-388 231-261 307-337
265 395 29 1 296 211 355
-
-
170-220 170-220 -
-
286 303 -
-
-
262-362 297-317 290-320 272-302 299-319
282 256 280 219 327
-
-
273-303
-
249
235-275 283-323 238-278 326-366 256-296 329-369 253-293 366-406 467-507 -
-
279 290 258 297 229 413 356 548 379 435 453
-
m-rn
9457.
222-262 296-336 378-408 380-420 235-265 258-288 222-262 -
222 274 480 465 299 246 212 -
335-475 435-475 280-395 282-322 -
316-356 170-220 170-220 345-495 345-495 345-495 316-356 385-425 260-300 255-295 305-345
431 547 274 268 318 285 222 599 453 504 317 368 233 251 417
430
KESSLER ET AL.
Table 1 (Continued)
SDS Proton I Phe7-HBSR Phe7-H"' Phe7-HBSR GlyS-NH Gly8-NH Giy8G ~ ~ ~ - H ~ ~ 1 ~ ~ G ~ ~ ~ - H ~ . ~ Prog-H" Prog-H" Prog-H* P ~ ~ ~ - H ~ , ~ POrg-H"' P~o~-H~,~ pro9-H6*s Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-NH Phe'O-H" Phe'O-H" Phe'O-H" Phelo-HA*S p he'o- H@sR Phe'o-H"' Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu(S)''-H" Abu(S) -Ha Abu(,)"-He Abu(S)"-HB Abu(S)" -HY Phe'*-NH Phe'*-NH Phe'2-NH Phe'I-NH Phe'*-NH Phe'2-H"' Phe'2-H"R ValL3-NH Val13-NH Val"-NH ValI3-NH ValL3-H" Val13-H" ValI3-H" Val13-H" Va1I3-H"
Proton 2
~
~
HzO
NOE
MD
NOE
MD
290-320 294-384 310-400 202-252 219-269 -
374 475 338 292 236 244 297 354 419 311 284 293 225 250 45 1 728 441 -
342-382
353 277 279 322 290 226 291 -
194-234 262-292 321-351 350-460 350-460 -
280-310 237-267 263-293 290-320 255-365 400-600 400-600 317-357 251-281 289-329 350-460 298-328 215-275 210-240 250-360 330-370 306-336 266-356 272-292 242-272 213-243 205-235 323-423 266-296 270-300 267-287 215-245 321-421 224-324 235-255 228-338 200-310 200-230 -
342 311 346 485 212 288
-
246 379 332 415 344 290 306 248 226 402 294 319 275 242 431 277 23 1 304 379 212 -
-
271-311 290-310 291-331 246-286 278-318 231-271 272-312 398-438 402-442 323-443 355-475 275-365 275-365
-
303 453 400 477 516 372 332 -
-
-
-
252 281 302 363 279 363 330 327 226 293 -
275-315 275-315 244-314 289-329 229-269
-
345-385 235-355 245-285 224-264 242-362 233-273 338-378 335-353 285-310 250-290 285-325 275-415 275-415 195-245 375-445
246 400 315 284 303 260 343 311 235 447
STRUCTURE OF Ro 09-0198
Table I
431
(Continued) SDS
Proton I Val13-H" Val13-H" Val13-HB va113-~73
Val'3-HY,R Ala(S)14-NH Ala(S)14-NH Ala(S)I4-NH Ala(S)14-NH Ala(S)14-Ha Ala(S)14-HB3s Ala(S)14-HB*R A~P-NH Asp15-NH Asp"-NH Asp15-NH Asp"-NH Asp"-H" G ~ ~ - N H Gly"-NH G~~~G-H".S
G~~~G-H*,R
G~~~G-H",R Asn17-NH AsnI7-NH Asn17-H" Asn17-H" Asn17-HB Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu(S)"-NH Abu ( S )''-Ha Abu(S)"-H" Abu ( S )''-Ha Abu(S)"-H4 Lyslg-NH Lyslg-NH Lyslg-NH Lyslg-NH Lyslg-NH LysIg-NH Lyslg-H" Lyslg-H" Lyslg-H" Lyslg-H"
Proton 2 Ala(S)14-HPsS Ala(S)14-HB9R Ala(S)14-NH Ala(S)14-NH Ala(S)14-NH Ala(S)14-Ha Ala(S)14-HB,S Ala(S)'4-H@3R Asp"-NH Asp"-NH Asp"-NH Asp15-NH Asp15-H" Asp15-H@ GlylG-NH G~~~G-H".s GlylG-H".R G1y1'-NH G~~~G-H".S Asn17-NH Asn17-NH Lyslg-Hy-S Asn17-H" As~'~-H@ Abu(S)"-NH Lyslg-NH Abu(S)"-NH Abu(S)l'-H" Abu(S)"-H@ Abu(S)18-Hy Lyslg-NH Lys'9-HO.R Abu(S)"-HB Abu(S)18-Hy Lyslg-NH Lyslg-NH Lyslg-H" Lyslg-H@,S Lys'g-H@,R Lys'g-Hy,S Lyslg_Hy,R Lys'9-H Lys'g-H@,s
Lys'g-H@,R Lys'g-Hy,S Lys"-H6
H20
NOE
MD
NOE
MD
-
-
250-350 268-398 269-289 240-290 250-300 222-252 316-366 346-392 290-310 342-372 250-280
352 497 288 304 256
442-472 372-412 452-492 -
530 401 43 1 -
-
335-365 280-310 285-305 301-321 -
270-290 220-340 208-248 250-380 300-460 270-310 337-367 266-376 234-264 395-425 230-255 231-291 -
258-278 270-300 -
215-245 233-263 400-500 219-249 234-264 270-300 380-480
-
208 373 426 284 394 252
-
323 262
-
340 224 291 306 221 392 392 285 354 356 244 459 245 278 274 347 219 321 560 256 304 313 455
-
-
272-312 210-250 231-271 348-388 204-254 380-420 395-435 285-310 321-361 355-405 395-465 415-445 255-295 245-285 415-455 293-313 241-351 219-259 279-389 339-379 258-378 225-265 263-303 285-405 314-354 328-368 273-319 295-335 226-266 326-336 -
270 329 195 43 1 195 374 448 285 385
-
446 477
-
-
525 321 216 462 292 316 242 334
-
355 291 269 200 303 350 398 279 355 234 278 -
-
-
231-271 294-334 -
259 302 -
-
432
KESSLER E T AL.
These distances, which are listed in Table I, were used in model building and restrained molecular dynamics (MD) for the structure determination.
MD CALCULATIONS All calculations were done with programs of the GROMOS ( GROingen Molecular Simulation) library5 on Silicon Graphics 4D/240SX and 4D/ 70GTB computers. A simulated annealing approach together with energy minimization ( E M ) was used to achieve preliminary structures from the given NOE data sets. The starting structure was taken from previous calculations of the structure in DMSO
s ~ l u t i o nFor . ~ ~both ~ calculations (H20 and SDS/ H20)we used the following scheme: -10 ps of high temperature MD at 1000 K with a force constant for the distance restraining potential of K h = 4000 kJ /mol- nm2. -10 ps MD for cooling down to 300 K and following EM. The preliminary structure in SDS /H20that results from MD calculations with 152 distance constraints (49 intraresidual, 38 sequential, 65 long range) differs in various aspects from the structure in H 2 0 (138 distance restraints, 41 intraresidual, 31 sequential, 66 long range). A comparison of the back-
Figure 1. Backbone atoms and sulfur bridges of cinnamycin. The structure on the left was obtained from NOEs measured in SDS/H20, the structure on the right from NOEs measured in HzO. The N- and C-terminal ends are oriented to the bottom. Nitrogens are stippled, oxygens and sulfur atoms are solid. The carboxyl group at the C-terminus is omitted for clarity.
STRUCTURE OF Ro 09-0198
433
STRUCTURE IN H20 AND SDS MICELLES
A Phe Harom /CHQ SDS
11
2
1
Phe Haom/ (CH2In SDS
Phe Harem/ HP
J
Phe Harem /(CHz-O-) SDS
I;
An inspection of the NOE table indicates that there is a change in the conformation when the environment is changed. A comparison of the backbone atoms of the result of restrained MD in vacuo using the experimental restraints as obtained in H20 and SDS micelles shows that the main difference is in a hinge region, which connects the hydrophilic part with the lipophilic one. The general structure of the @-sheetof the A ring (amino acids 1-4 and 14-19) is retained but the hydrogen-bond pattern has changed. It should be noted that the results have to be refined by MD calculations including the solvent. Our preliminary results indicate that in spite of the polycyclic skeleton there is still a lot of flexibility in the structure.
ORIENTATION IN MEMBRANES
Phe Harem /Ha
(NOE,exchange)
NH2’Hz0
l5
Gln3,Asn17 NH2 (NOE,exchange)
The amphiphilic nature of cinnamycin immediately led to the speculation of its orientation in a biological membrane. For a proof of this we have recorded proton-MAS spectra of cinnamycin in SDS bilayers. A NOESY spectrum under these conditions contains a set of highly resolved cross-peaks (Figure 2 ) between the aromatic protons of cinnamycin and the CH, groups of SDS lipophilic chains. This provides evidence that the lipophilic end of the peptide sticks in the lipophilic part of the bilayer. Financial support by the Deutsche Forschungsgemeinschaft and the Fonds der chemischen Industrie is gratefully acknowledged.
REFERENCES
ppm
7
6
Figure 2. Part of the 500-MHz ‘H MAS NOESY spectrum of cinnamycin in SDS bilayers. In F2 only the region of the NH and aromatic protons is shown. Measurement conditions: SDS-DIZ (25 wt% ) , SDS-HI, (6.5 wt% ) , cinnamycin (13.6 wt% ), H 2 0 (54.4 wt%) at 297 K, molar ratio SDS/cinnamycin = 18; mixing time = 100 ms. A jump return (JR) observing pulse in the NOESY sequence and 5-kHz MAS were used.
bone atoms is shown in Figure 1. Calculations in the corresponding environments are in progress and should lead to more refined structures.
1. Wakamiya, T., Fukase, K., Naruse, N., Konishi, M. & Shiba, T. (1988) Tetruhed. Lett. 29,4771-4772. 2. Wiltschek, R. (1991) Diplomarbeit, Prof. E. Sackmann, Technische Universitat Munchen. 3. Hyberts, S. G. & Wagner, G. (1989) J. Mugn. Reson. 81,418-422. 4. Kessler, H., Seip, S. & Saulitis, J. (1991) J. Biomol. N M R 1,83-92. 5. van Gunsteren, W. F. & Berendsen, H. J. C. (1987) Groningen Molecular Simulation (GROMOS), Library Manual, Biomos, Groningen. 6. Will, M. (1989) Ph.D. thesis, Frankfurt/Main. 7. Kessler, H., Steuernagel, S., Will, M., Jung, G., Gillessen, D. & Kamiyama, T. (1988) Helv. Chim. Actu 71,1924-1929.
Received July 1 , 1991 Accepted August 13, 1991
