The structure and conformation of HC-toxin

Vol. 111, No. 2, 1983 March

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

16, 1983

Pages

398-403

THE STRUCTURE AND CONFORMATION OF HC-TOXIN Megumi

Kawai

and Daniel

H. Rich*

School of Pharmacy University of Wisconsin-Madison Madison, Wisconsin 53706 Jonathan Department

of Plant Cornell

Breeding and Biometry University New York 14853

Ithaca, Received

January

D. Walton*

14, 1983

Difference nuclear magnetic resonance studies experiments establish the sequence and configuration HC-toxin toxin as cyclo(L-Aoe-D-Pro-L-Ala-D-Ala). conformation in solution previously found for the peptide chlamydocin. The host-specific Helminthosporium

toxin,

carbonum

considerable

attention

structural (I)

cycle

decanoic

chlamydocin

(7).]

cyclic

While magnetic the

acid,

amino

acid

et al.

(5).

amino

acid

a novel

differ

a cytostatic

resonance

*To whom correspondence

of amino

and chirality are

cyclic the

closely

is

solution

cycle

should

on maize

culture

filtrates (1)

(2,3)

has attracted

and its

have been proposed et al. [Aoe

(4)

previously acids

for

in both

unusual for cycle

in CyL-2

structures only

to the

HC(L-

2-amino-8-oxo-

found

was proposed related

and (II)

stands

of

for

system

(6)

are the II. found

Both in

tetrapeptide.

(NMR) spectroscopy,

sequence

1 by Pringle

by Liesch

structures

investigating

race

two structures

The composition

sequences

from

effects

(Aoe-Ala-Ala-Pro),

tetrapeptide

chlamydocin,

its

by Walton

9,10-epoxy

same but the

for

Recently

Aoe-D-Pro-L-Ala-L-Ala)

isolated

(cochliobolus)

both

properties.

toxin:

HC-toxin,

and amino acid oxidase of amino acids in HCadopts the bis-y-turn cytostatic cyclic tetra-

conformation we obtained

(Aoe-D-Pro-Ala-Ala) be addressed.

0006-291X/83 $1.50 Copyright 0 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.

398

of HC-toxin evidence (II),

by nuclear confirming

but

consistent

that

and

Vol. 111, No. 2, 1983

with

the

report the

second

here

the

sequence

tion

alanine

residue

results

of the

and chirality

of HC-toxin

Materials

BIOCHEMICAL

AND BIOPHYSICAL

having

the

0 - not the

NMR and amino

of amino

acids

RESEARCH COMMUNICATIONS

acid

L - configuration.

oxidase

and establish

studies the

We

that

overall

confirm

conforma-

in chloroform.

and Methods

The isolation and the purification of HC-toxin has been reported in detail (5). Chloroform-d (99.6% atom XD) was purchased from Aldrich Chemical co. Proton NMR spectra were recorded on a Bruker WH-270 (270 MHz) spectroDetailed descriptions of experimental meter operated in the FT mode at 30°C. methods for the NMR studies are reported in preceding papers (8,9,10). The carbon-13 NMR spectrum was recorded on a Bruker WM-300 (75.5 M Hz) at 25'C. The amino acid oxidase experiments were carried out essentially by the procedure of Closse and Huguenin (5) as reported for chlamydocin. HC-toxin (1 12 hr), and the mixture treated sepapmole) was hydrolyzed (6 N HCl, llO”, rately with L-amino acid oxidase and D-amino acid oxidase as described. The reaction was stopped by boiling, the solution was filtered and the filtrate loaded directly onto the Beckman column. Amino acid analysis after digestion with L-amino acid oxidase showed 22.7 nmole Ala:2.3 nmole Pro and after reaction with D-amino acid oxidase, 27.7 nmole Ala:27.3 nmole Pro, respectively.

Results The 270 MHz lH NMR data reasonably and the

well coupling

with

are

reported

constant Table

'H NMR data

values 1.

1: 2:

(d, 1 (d, 1 (d, 1 (m, 1 (d.d, (m, 1 (m, 1 (m, 1 (m, 1 (d-d, (d.d, (d.d, (m, 6 (m, 8 (d, 3 (d, 3

are

slightly

in Table (4,5)

1.

although

These the

different.

results chemical

The two

Ii, J = H, J = H, J = H, Aoe 1 H, J H, Ala4 H, Ala3 H, Pro H, Pro 1 H, J 1 H, J 1 H, J H, Pro H, Aoe H, J = H, J =

- D-Pro2-L-Ala3-D-Ala43

10.3 Hz, Ala3 NH) 10.5 Hz, NH) 9.5 Hz, Ala Aog NH) a-CH) = 1.6, 6.0 Hz, Pro a-CH) a-CH) a-CH) 6-CH) 6'-CH) = 2.4, 4.6 Hz, Aoe epoxy") = 4.7, 5.8 Hz, Aoe epoxy') = 2.4, 5.8 Hz, Aoe epoxy' ) 6, y, Aoe U) s, y, 6, E) 6.8 Hz, Ala3 s-CH ) 7.0 Hz, Ala4 s-CH:)

Peptide concentration: 6.8 mg/0.35 mL CDC13 at 30°C. Temperature range 30-56.6"C: x 1O-3 ppm/“C 399

agree shifts

alanine

270 MHz lH NMR data for K-toxin1 [L-Aoel

7.12 6.32 6.25 4.77 4.70 4.56 4.46 3.97 3.52 3.42 2.99 2.86 2.40-2.48 1.55-1.97 1.32 1.27

summarized

-1.73 -2.97 -5.00

Vol. 111, No. 2, 1983

BIOCHEMICAL

Table

2. Results

AND BIOPHYSICAL

of NOE experiments

RESEARCH COMMUNICATIONS

of HC-toxin

in chloroform-d irradiate

signal

Ala3 Aoe Ala4 Aoe Pro Ala4 Ala3

residues

were

Overhauser

for

assigned

experiments

peptide

Table

Pro Ala4 Ala3 Pro Ala3 Aoe Ala4

by standard

double

(Table

Large

2).

have also

12) suggesting

transoid

observed

NH (7.12) NH (6.32) NH (6.25) a-CH (4.77) a-CH (4.70) a-CH (4.56) a-CH (4.46)

NH and Aoe NH protons (8,9,11,

(ppm)

that bonds

(13).

3. Carbon-13

coupling

shifts

chlamydocin

of HC-toxin

polypeptide

a

57.98 25.06 24.96

Aoe a chain

Epoxy a 6

51.98 29.01, 25.45 22.75 36.26

28.68

53.36 46.03

of the

and

c=o

54.39 28.77 25.30 22.86 36.30 53.36 45.99

Aib a f3 6' Ala a %

NH) for

ea, NH values

57.86 25.03 24.76 47.02

47.19

58.89 26.55 23.57

48.21, 14.58,

47.58 14.00

207.51

(Aoe epoxy)

173.81 173.75 173.36 171.44

207.27 175.63 174.33 172.87

(Aoe)

171.84

in Ref. 9 400

nuclear

Ala3

systems,

53.52 35.92

6 Y 6

a cited

(3Ja,

by

chlamydocina

B

side

and

in chloroform-d

phe a pro

:::

coefficients

HC-toxin

carbon

< 1.0 10.1

constants

in other

The temperature

13.3 13.6 9.0

experiments

NH 2 10 Hz represents

chemical

%

a-CH (4.70) a-CH (4.56) a-CH (4.46) a-CH (3.97) NH (7.12) NH (6.32) NH (6.25)

resonance

been observed

3Ja,

NOE (ppm)

= 180' amide

BlOCHEMlCAl

Vol. 111, No. 2, 1983

protons

(Table

solvent,

1) establish

consistent

with

The Cl3 NMR data of chlamydocin

(9).

carbons

the

except

25.06

in

trans

preceding

found but

two

for

the

L-Ala

able

and part

in chlamydocin

establishing

all

with

chemical

shifts

difference

In HC-toxin at 54.39

the

x-Pro

for

the

reasons

between

this

carbon

those of

resonate

that

y-turn

from

compared

The Pro CB and Ca carbons

One major

resonates

shielded

(9).

3, are

to assign

of an inverse

Aoe a-carbons.

Aoe NH are bonds

in Table

we were

carbons.

(8,9).

hydrogen

listed

ppm respectively,

(14)

RESEARCH COMMUNICATIONS

NH and the

intramolecular

HC-toxin,

carbonyl

papers

at the

that

By analogy

ppm and 24.96

dominantly

AND BIOPHYSICAL

bond

the two

resonates

at is

pre-

discussed

compounds at 51.98

is

ppm,

ppm.

Discussion To distinguish we carried

between

out difference

chlamydocin

(8,9)

is

when the

expected

showed

that

a strong

(4.70

is tent

found

in

with

resonates in the

II

this

and also

is

2.4

13C NMR.

This

sequence

and the for

The difference reassigned rotations

from about

finding from

shift

Aoe-Ca

of the is

must

the is

amide

irradiated.

at Pro-d

ppm produced is

located

in HC-toxin in chlamydocin

by steric

an enhancement

near

be Ala-L-Aoe

also

Since

as

Consis-

position

caused

Aoe-

D-Fro-L-Ala-

Aoe a-carbon

not

the

Ala4

compression

suggested

the

all

observed

or about

the 401

3Ja,

of Ala4

a-H, the

a D-Pro-Aoe.

be (L-Aoe-D-Pro-L-Ala-Ala)

NOE data

of Pro-d

when the

sequence

corresponding

probably

Our results

bond configuration.

the

with

of D-proline

a 13% enhancement

partial

that

a-proton

HC-toxin

and Pro-C&.

must

bonds

results

residue

a trans

is the

sequence

amide

previous

the

establish

Aoe NH proton

L to D.

on our

data

upfield

HC-toxin

for

was observed

of Aoe NH at 6.32 the

next

proposed

No enhancement

establish

the

Based

and II)

ppm) was irradiated

ppm upfield

l5 between

indicates trans,

(7.12

(I

enhancement

NH of the

assignment

Irradiation This

amide

These

about

(v-effect)

positive

ppm).

NH was irradiated.

two sequences

NOE experiments.

when AlaI-NH

was obtained

the

$J, + torsion

angles

bond

(5).

of Ala4

NH values

amide

Thus the

as in II chirality

a-H.

are are

should

large,

be

rapid

not expected.

Vol. 111, No. 2, 1983

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Schematic drawing of sequence and chloroform Dashed lines indicate probable of HC-toxin.

Figure 1.

solution hydrogen

conformation bonds.

Torsion angles (+ 20") are: L-Aoe, @,-l20; 9, 120; o, -160. D-Pro, L-Ala, 4. -110; $, 110; o, -160. D-Ala, 9, 60; 9. -55; w, tl60. 4. 60; JI. -50; w, 160.

The observed

coupling

NOE enhancement amino

acid.

between

between

enhancement

also

HC-toxin and D-amino

establishes

acid

as predicted

HC-toxin

is

cycle

In both established

from

the

if

3Ja, NH would would

Ala3

with

presence

NMR data.

Thus,

L-amino

The NOE

acid

of alanine

sequence

oxidase

was oxi-

and 1 D-Ala

in

and chirality

The chirality

(L-Aoe-D-Pro-L-Ala-D-Ala).

a D

and Ala4.

of 1 L-Ala the

is

be - 6 Hz and no NOE

one equivalent

the

Ala4

positive

be expected.

bond between separately

cases

NH and the

of the

of

epoxide

has not been assigned. Our data

very

tides

amide

Ala4

reasonable

a-CH protons

and reacted

enzyme which

Hz) for

NH is

at Ala4

a trans

oxidase.

HC-toxin

is

a-H and Ala4

Aoe NH and Ala4

was hydrolyzed

by the

group

Ala3

(3J NH = 9.5

For an L-configuration

enhancement

dized

constant

also

similar (8).

to that

The temperature protons

that

establish

establish dependencies are

the

conformation

of chlamydocin

The NOE data

The 13C NMR data

Aoe amide

establish

likely

the

(9)

that

of the

and its

HC-toxin

D-Pro-L-Ala amide

to be hydrogen

402

of HC-toxin related has four

sequence protons bonded.

part

in chloroform

cyclic trans

tetrapepamide

bonds.

of an inverse

establish Together

both

the

these

y-turn. L-Ala data

and are

Vol. 111, No. 2, 1983

consistent

with

The fully noted are

the

bis-y-turn

ring

to uncover

conformation

of the Both

similar. systems

AND BIOPHYSICAL

NMR results

structures

remarkably

Attempts

the

characterized

that

peptide

BIOCHEMICAL

adopt the

will

the

bis-y-turn

respective

schematically

be reported

cytostatic

contain

the

shown

RESEARCH COMMUNICATIONS

separately. chlamydocin

Aoe residue

and both

of action

Figure It

compound

conformation

sites

in

should

be

and HC-toxin cyclic

in chloroform for

1.

tetrasolution.

these

molecules

by Grants

AM20100

are

in

progress.

This

Acknowledgements. CA27985 Grant

from the #79049

work

was supported

National

Institutes

to Elizabeth

D. Earle,

in

of Health Department

part

and

and by Rockefeller

Foundation

of Plant

Cornell

Breeding,

University.

References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15.

Pringle, R. B. Plant Physiol. (1970),%, 45-49. Pringle, R. B. Plant Physiol. (1972), 48, 756-759, Scheffer, R. P. and Ullstrup, A. J. Phytopathology (1965),%, 10371038. Liesch, J. M., Sweeley, C. C., Staffeld, G. D., Anderson, M. S., Webber, 0. J. and Scheffer, R. P. Tetrahedron (1982), 38, 45-48. Walton, J. D., Earle, E. D. and Gibson, B. W. Biochem. Biophys. Res. Commun. (1982), 107, 785-794. Hirota, A., SuzuK A., Aizawa, K. and Tamura, S. Agr. Biol. Chem. (1973), 37, 955-956. Closse,7f; and Huguenin, R. Helv. Chim. Acta. (19741, 57, 533-545. Rich, 0. H. and Jasensky, R. D. J. Am. Chem. Sot. (198v, 102, 11121119. Rich, D. H., Kawai, M. and Jasensky, R. D. Int. J. Peptide Protein Res., in press. Kawai, M., Jasensky, R. D. and Rich, D. H., submitted for publication. Wagner, 6. Dissertation, E.T.H., Zurich, 1977. Llinas, M., Wilson, D. M. and Neilands, J. B. Biochemistry (1973),x, 3836. Demarco, A., Llinas, M. and Wuthrich, K. Riopolymers (1978), 17, 637. Deber, C., Madison, V. and Blout, E. R. Act. Chem. Res. (1976x2, 106113. Jager, Y. and RUB, V. Liebigs Ann. Chem. (1980), 101-121.

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