1H n.m.r. studies of a neurotoxin and a cardiotoxin from Naja mossambica mossambica : Amide proton resonances

Vol. 76, No. 4, 1977

H n.m.r.

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

STUDIES OF A NEUROTOXIN AND A CARDIOTOXIN sambica:

FROM Naja mossambica

AMIDE PROTON RESONANCES

Jiirgen Lauterwein, Kurt Wiithrich, Hugues Schweitz*, Jean-Pierre Michel Lazdunskif Institut fiir Molekularbiologie und Biophysik, Technische Hochschule, CH-8093 Ziirich, Switzerland, and *Faculte Universitd de Nice, Part Valrose, F-06034 Nice, France Received

April

mos-

Vincent* and Eidgendssische des Sciences,

25,1977

SUMMARY: Proton n.m.r. spectra at 360 MHz of neurotoxin II and cardiotoxin V II 4 from the venom of Naja mossambica mossambica are reported. From the n.m.r. spectra the solution conformations of the two proteins seem to be quite closely related. However, the exchange rates of the n.m.r. observable labile protons with deuterium of the solvent were markedly different, showing that the molecular structure of the cardiotoxin must be more flexible than that of the neurotoxin and suggesting that the different functional properties of the two toxins might be related to the different molecular dynamics.

Snake neurotoxins

and cardiotoxins

homologies

(l-5)

chain

60 - 62

with

half-cystine

. Short

positions,

groups

in cardiotoxins

differences

in

to a protein

receptor Cardiotoxin

to trigger K+-ATPase

(5). study

mossambica the

structural

other

amino

In spite

acid

bridges.

level

of the neurotoxin

and blocks

mossambica,

which for

and cardiotoxin

was undertaken

the different

V

to obtain

functional

at the same found

there

of toxins.

that

Neurotoxin

trans-

structure, the Na+,

inactivates

4 from

resonance the snake -

additional

properties

binds

acetylcholine

magnetic II

to be com-

are pronounced

receptor

in the membrane 1 reports a H nuclear

II

are

homologies

rearrangement paper

The eight

are placed

to a lipid-type

structural

of one polypeptide

residues

two types

binds

extensive

consist

disulfide

of these

of the

apparently

The present

basis

four

at the post-synaptic

a structural

with

and neurotoxins

the mode of action

mission

(6).

seven

of toxins.

proteins

and cardiotoxins including

acids,

and in addition

mon to both

(n.m.r.)

neurotoxins

amino

residues

are parent

insights of

into

the two pro-

teins. II MATERIALS AND METHODS: Neurotoxin II and cardiotoxin V 4 from the venom of Naja mossambica mossambica were prepared as described previously (5,7). For the n.m.r. measurements, 0.002-M solutions of the proteins in D20 at pD.v4.5 and which contained also 0.1-M NaCl, were prepared. pD values are the pH PD -7.0, meter readings without correction for isotope effects. High resolution proton n.m.r. spectra were obtained on a Bruker HX-360 spectrometer. Chemical shifts are in parts per million (ppm) relative to internal sodium 3-trimethylsilyl propionate (TSP).

Copyright All rights

0 1977 by Academic of reproduction in any

Press. Inc. form reserved.

1071 ISSN

0006-291

X

Vol. 76, No. 4, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

cardiotoxin

I

I

I

I

IO

I

8

I

1

I

6

I

I

4

1

I

2

0

PPM Fig.

1

-3 H n.m.r. spectra at 360 MHz of 2.10 -M solutions of two toxins from Ne in D20, which also contained 0.1 M NaCl, T = 25v. The neurotoxin solution had pD = 4.8, the cardiotoxin solution pD = 4.1. Both spectra were recorded within 15 min after the proteins had been dissolved in D201

RESULTS: Fig. cardiotoxin dition

recorded to the

aromatic from

1 shows the

7 to

10

ly exchanging

immediately

resonances

protons

360 MHz 'Ii

between

after

dissolving

of the aliphatic

protons

6 and 8 ppm (B),

these

ppm, numerous labile

spectra

n.m.r.

protons

quite

well

resolved

in the protein.

1072

of

the

neurotoxin

the proteins between spectra lines

in D20.

In ad-

0 and 6 ppm and the contain,

which

The present

and the

in

correspond investigation

the range to

slow-

concen-

Vol. 76, No. 4, 1977

trated

on the

determine

BIOCHEMICAL

spectral

features

of these

the number

of slowly

exchanging

sure

their

rium

of the solvent.

chemical

shifts

spectra

of

formations

of the neurotoxins

range

at least

from

the 1H n.m.r.

minary

observations

change

rates pH.

This

8.0

had also

is

amide

we decided

prepare

protein

overall

be rather

on

be studied

within

The proteins

typical

for

changes

qualitative

at pDor 4.5 and then

even

dissolved

time

that

the

1) of these intervals actual

toxin.

From comparison

labile

protons

proteins

of these

7.02

to 9.89

rived

from

ter

at pD = 4.1 less After

than

course than

with

1 hr

details

(Table

also

As a general

for

first

exchange

to

would

at higher protons

pD could

in H20 and the pH adjusted

were

from I).

these 7.55

complete

to 9.84

The lyo-

ca.

tubes for

protons.

of the of both

The chemical

30 protons

rates

de-

considerably

In the neurotoxin 1 hr.

and from

exchange

labile

hours showed

the cardio-

exchange

were

30 observable than

several

the spectra

the proton

I).

spec-

ppm in neurotoxin

intensities

of less of the

and 4.1

30 labile

Overall,

ca.

during

in the n.m.r.

conditions

(Table

among the

lyophilized.

recorded

after

of the resonance

in neurotoxin

then

neurotoxin

of approximately

a half-time

and in

ml of D20 at 25O, and n.m.r.

the

under

(Table

at

(10-12).

studies

were

obtained

range

cardiotoxin

for

and T = 25O, thirteen

the proton

25O (for

resonances

and T = 25O, eight

to exchange

that

(9),

exchanging

pH measurements

spectra

resonances

the time

in cardiotoxin

pD = 4.8 found

in

with

solutions in 0.4

4.8

the ex-

flexibility

proton

slowly

D20 solutions

were

was found

contained

shifts

0.002-M

the pH

of time.

These

of 15 minutes. pD values

it

the most

con-

45O. Preli-

least

at

4.0 to 8.0

the kinetic

in 01-M NaCl solution

redissolved

(Fig.

pursue

over

observations,

and 25O , where

period

philized tra

to

the molecular

in general

of the molecular

of these

deute-

7 and 10 ppm decrease

protons

the basis

of HCl.

with

amide

to

to mea-

with

maintained

up to

proteins

where

were

were

between

rates

that

in the pH region

observed

to 4.0 by the addition proteins

that

protons

a reasonable

were

1) had shown

was used

two proteins,

exchange

in globular

slow,

temperature,

the proton

at temperatures

and

N.m.r.

in the

protons

solutions

and higher

protons

(Fig.

shown

of pH dependent

exchanging

strategy

to

behavior

protons.

and the cardiotoxin

of the labile

the absence slowly

4.0

labile

and to study

Inspection

lower

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

fas-

solution protons

In cardiotoxin had a half

at

were solutions

time

of

I).

exchange see figure

had been captions

followed

for

2 and 3),

1073

several the

hours

temperature

at pDq4.5 was raised

and to

Vol. 76, No. 4, 1977

Table

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I Chemical shifts,d,and life times with respect to chemical of the slowly exchanging labile protons in neurotoxin II toxin VII4 of Naja InOssambica mossambica. Neurotoxin

r1 T

7.55 7.50 7.62 7.98 a.05 8.28 8.60

hr - 10 hr at 35O and pD = 6.2

10 hr

- 100 hr

7.64 7.94 a.17 a.52 9.09 9.43 9.65

> 100 hr at 35O and pD = 6.2

a For resonances mated resonance

phosphate thus

buffer.

obtained

in both

protein

(2ja

7.21 7.46 7.51 8.58 8.84 a.94 9.02 9.16 9.24 9.44 9.60

(2-3ja (21a

(aa

8.16 a.58 9.89

i2-3ja

corresponding to more than indicated in parentheses.

by the addition

pH measurement

7.02 7.51 7.60 7.67 7.77 7.85 7.% a.21 a.31 a.39 8.86 9.25 9.38

7.96 a.87 9.41 9.84

with intensities intensities are

35O and the pD changed

6 (ppm)

1.44 7.89 8.65 a.72 8.87 a.95 Q-OS 9.09 9.33 9.53

at 35O and pD = 6.2

T

Cardiotoxin

G(pprn)

0.1 hr - 1 hr at 25O and pD = 4.8 (neurotoxin) or pD = 4.1 (cardiotoxin)

0.1

II

exchange, and cardio-

of 0.01

in the n.m.r. solutions.

ml of 2-M deuterated tubes

The time

1074

one proton,

showed

course

that

of the

esti-

neutral pD = 6.2 was resonance

in-

P

Vol. 76, No. 4, 1977

BIOCHEMICAL

lo Fig.

2

Fig.

at pD = 6.2

3 for

exchange

PPM

and

cardiotoxin.

is

with

DISCUSSION: the neurotoxin

of 100 labile

= 35' the

faster

deuterium

In all,

T

Again

appreciably

2 and 3 are presented

der

8

Amide proton exchange in a D20 solution of neut;otoxin II. The sample of Fig. 1 had been kept at pD ; 4.8 and T = 25 for 5.5 hrs. The temperature was then raised to 35 and the pH changed to 6.2 by the addition of a trace of deuterated 2-M neutral phosphate buffer. The spectra were recorded at the following time intervals after the addition of the phosphate buffer: A. 16 min, B. 17 hrs, C. 73 hrs, D. 454 hrs.

tensities change

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

is

shown in Fig.

two figures

in Table

protons

cardiotoxin per

neurotoxin

show that

Life

times

from experiments

and in the proton

with

of the

respect type

exto

of Figs.

I.

the polypeptide

and the

clearly

in cardiotoxin.

of D20 estimated

2 for

backbone

and the

used in the present

molecule.

The majority 1075

amino

acid

study of these

side

contain protons,

chains

of

of the ori.e.

Vol. 76, No. 4, 1977

lo

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6

8

PPM

Fig.

3 Amide proton exchange in Fig. 1 had been kept at ture was then raised to Fig. 2. The spectra were vals after the addition C. 3.6 hrs, D. 53 hrs.

a D20 solution of zardiotoxin. The sample of pD = 4.1 and T = 25 for 3 hrs. The tempera3S" and the pH changed to 6.2 as described in then recorded at the following time interof the phosphate buffer: A. 10 min, B. 32 min,

those

of the

backbone

chain

protons,

D20 solutions teins in

would

the spectra

solvent

accessible

exchange

too rapidly

of the proteins therefore of Fig.

suggest

(8,9). that

amide

groups

to be observed General

experience

essentially

1 to 3 come from backbone

1076

in

all

and the bulk the n.m.r.

of the side spectra

of

with

peptides

and pro-

the labile

protons

observed

located

in inte-

amide

groups

Vol. 76, No. 4, 1977

rior

parts

(8-12).

BIOCHEMICAL

of the molecule

This

multiplet

is

further

(a-12),

a suitable

and hence

not

corroborated

structures

cussed

AND BIOPHYSICAL

method

accessible

by the chemical

of the resonance

studies

readily

RESEARCH COMMUNICATIONS

lines.

of the exchange

to investigate

shifts

(Table

As was previously

kinetics

the

to the solvent

of interior

rigidity

I)

and the

extensively amide

of the protein

dis-

protons

are

conformation

in

structures

of

solution. specific

More

resonance

homologous sheets ber

neurotoxins

containing

of slowly

number neurotoxin

exchanging

crystal

compatible

with

that

milar

to the

sults

of an earlier

side

crystal

mainly

of the

ticular

interest

protons

observable

neurotoxin

the spatial amide

neurotoxin. lecules

exchange

In other to occur

In conclusion

in

these

different

modes of

ty of the

spatial

possible that

it

changes

mechanistic is

a priori

are induced

(Table

I),

of cardiotoxin (13-15).

Considering

words, "open

should rates

action

also

related.

is

forms"

comparative

(l-51,

a greater

from which studies

structures

related

of neurotoxin

implications more likely

are

of this for

by the different

the structure

type

homologies it

is

not

in the

unexpected

On the other

hand, than

for

cardiotoxin

can take

place

(g-12).

that

to the different

flexibili-

hypothesis, molecule

it

Without

the

pursuing

may be pointed that

conformational

receptor

sites,

in mo-

seem to indicate

medium in or near

1077

of lato be that

and cardiotoxin.

a flexible

numbers

indicate

probability thus

acid

of par-

in cardiotoxin

NH-exchange

would

on membranes

faster

is

it

slowly

the extensive

considerably

amino

which

and cardiotoxin.

data

with

be closely

si-

(15),

identical

and cardiotoxin

are

there

(5,6),

n.m.r.

coincides

is

by the re-

sufficiently

the present

is

seem to

solution

of the

of neurotoxin

exchange

in

was known.

essentially

shifts

the

bonds

neurotoxin

structure

features

of

data

supported

properties

two proteins

chemical

of neurotoxin

structures

proton

structural

in the

conformation

functional

in

nuclei

the X-ray

the

of the resonances

the n.m.r.

also

of non-labile

before

structure

conformation is

with

hydrogen

of a homologous

features

different

(81,

the num-

closely

of most

conclusion

investigation

similar

sequences

that

This

appeared

since

backbone

that

sheet

shifts

to compare

amino

B-pleated assignment

polypeptide

of B-pleated

I: coincides

chemical

suggested

for

acid

antiparallel

structure.

determined

striking

quite

the observed

in D20 solution

the molecular

is

in Table

Since

on spectral

with

It

regions

in intramolecular

n.m.r.

On the one hand,

bonds.

extended

involved

and which

In view

include

by the X-ray

protons

the neurotoxin

chains

bile

in the

amide

indicated

listed

conformation.

imply

was based

which

protons

bonds

I. to backbone

also

the

(13,141,

are

25 to 30 hydrogen

of hydrogen

Table

assignments

out which

Vol. 76, No. 4, 1977

might function

be amenable relations

BIOCHEMICAL

to experimental in these

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

observation

in

future

studies

of structure-

toxins.

ACKNOWLEDGEMENTS: Financial support by the Schweizerischer ject 3.1510.73) and by the Centre National de la Recherche gratefully acknowledged.

Nationalfonds Scientifique

(prois

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

Lee, C.Y. (1972) Annu.Rev.Pharmacol. 12, 265-286. Yang, C.C. (1974) Toxicon 3, l-43. Louw, A.I. (19741 Biochem.Biophys.Res.Commun. E, 1022-1029. Gregoire, J. and Rochat, H., Eur.J.Biochem., submitted for publication. Vincent, J.P., Schweitz, Y., Chicheportiche, R., Fosset, M., Balerna, M., Lenoir, M.C. and Lazdunski, M. (1976) Biochemistry Is, 1371-1375. Rang, H.P. (1975) Q,Rev.Biophys. I, 283-399. Chicheportiche, R., Vincent, J.P., Kopeyan, C., Schweitz, H. and Lazdunski, M. (1975) Biochemistry 14, 2081-2091. Wiithrich, K., NMR in Biological Research: Peptides and Proteins, North Holland, Amsterdam 1976. Englander, S.W., Downer, N.W. and Teitelbaum, H. (1972) Annu.Rev.Biochemistry g, 903-924. Hvidt, A. and Nielsen, S.O. (1966) Advan.Protein Chem. 2, 287-386. Masson, A. and Wiithrich, K. (1973) FEBS Lett. 31, 114-118. Wagner, G. (1977) Ph.D. Thesis, Eidgendssische Technische Hochschule Ziirich, Switzerland. Low, B.W., Preston, H.S., Sato, A., Rosen, L.S., Searl, J.E., Rudko, A-D. and Richardson, J.S. (1976) Proc.Natl.Acad.Sci. USA 73, 2991-2994. Tsernoglou, D. and Petsko, G.A. (1976) FEBS Lett. "";i-4. Arseniev, A.S., Balashova, T.A., Utkin, Y.N., Tsetlin, V.I., Bystrov, V.F., Ivanov, V.T. and Ovchinnikov, Y.A. (1976) Eur.J.Biochem. -71, 595-606.

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