- Email: [email protected]
Molecular conformation of erabutoxin b; Atomic coordinates at 2.5 Å resolution
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 13, 1979
Pages 950-959
MOLECULAR CONFORMATION OF ERABUTOXIN b; ATOMIC COORDINATES AT 2.5 i RESOLUTION M. R. Kimball, A. Sate*, Department of Biochemistry,
Received
April
J.
S. Richardson**, L. S. Rosen, and B. W. Lowi Columbia University, 630 W. 168 St.,N.Y.,N.Y.
10032
16,1979
determined from a 2.5 g electron density map are SUMMARY: Atomic coordinates given for erabutoxin b, a sea snake venom postsynaptic neurotoxin. The principal structural features, anti-parallel 8 pleated sheet, 8 bulge and 8 bends are described. The erabutoxin b structure is discussed as structural prototype of this class of homologous curare-mimetic neurotoxins from both land and sea snakes. In this sea snake
communication
we provide
venom neurotoxin,
based
major
features
single
chain
of the molecular protein
of nearly phid)
50 homologous
snake venans
membrane mimetic
distribution
neuromuscular
density
as prototype
from both
to the
erabutoxin
for
Laticauda land
cholinergic
of this
a whole
class
semifasciata,
(elapid) receptor
transmission
b, a
map and describe
and stereochemistry
considered
structural were
features described
of this
of is
and sea
one
(hydro-
at the postsynaptic
in a nondepolarizing
The significance
(1).
was recognized series,
and a reactive short
with
60+62
curare-
disulfide
linkages.
toxin
and a preliminary
of the site
chain
observed
at a
discussion
of the resi-
and conservatively-substituted+ erabutoxin
region
and long
and 7 conservatively-substituted
invariant
short
earlier,
of some common invariant
dues given
iant
w electron
from the sea snake
bind
for
mode.
resolution
toxin
on a 2.5
neurotoxins
which
and block
The main lower
b,
coordinates
conformation
(Mr 6861)
Erabutoxin
neurotoxins.
atomic
described with
71+74
positions These
b structure
latter
as prototype
(1,2).
There
are two
residues,
with
18 invar-
in common, which play
a unique
include
and evident
four struc-
*Present address: Department of Chemistry, Tohoku University, Sendai, Japan. **Permanent address: Department of Anatomy, Duke University, Durham, N.C. t To whom all correspondence should be addressed. +Conservative substitution is used here to define conservation of residue type, i.e., Ser/Thr-hydroxyl group, Glu/Asp-carboxyl group, Val/Leu/Arghydrophobic group and Tyr/His/Trp-aromatic group. 0006-291 Copyright All rights
X/79/1
10950-10$01.00/O
@ 1979 by Academic Press, Inc. of reproduction in anyform reserved.
950
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-$ Fig. (-1
1. Ramachandran and "outer limit"
Fig. 2. produced
tural they
role; are
Stereo drawing by the GRIP-75
both reduced
native
angle plot for erabutoxin b showing "fully allowed" (----I boundaries. CO1 Gly; [@I Other residues.
of erabutoxin b structure showing Molecular Graphics System.
conformation
and toxicity
(3).
951
are
lost
all
the
residues,
completely
when
Vol. 88, No. 3, 1979
BIOCHEMICAL
TABLE 1
I”& 101 AR‘ L 114 ::, 126 123 1
101
IOX
74 LO 96
93 100 I"2
CL m;
11. 164 165 115 ;::
103 95 100 99 106 92
97 b7 74 65 53 b7
c D
129 122
73 73
112 122
1*1 14Y 154 157 144 143 162 171
61 66 41 39 38 24 65 60
111 123 120 133 112 117 123 116
cc co bb
2
:‘a
L CB CG2 CGl co1 c ‘ 3
lb4 177 174
11 75 96
132 134 133
179
65
152
4
kbb 193 2Ul 216 222 221 22, 234 235 229 1YI 202
62 70 19 66 55 42 41 52
152 163 160 15b 164 161 i49 140 144 176 1,7
cl.
CL2 Ccl2 c 0 5
191 166 17? lb9
159 173 2"O 200
‘A CB ct CD U&l L‘2 c (r
165 198 205 215 212 227 206 220
212 i23 235 24, 254 246 26, 229 234
79
234 22, 233 239 233 242 251 252 247 253
100 90 9, 107 113 121 121 112 102 93
76
70 73 71 66 72 n9 96
203 211 202 20, 199 190 196 219 211
231 236 253 2b1
272 27, 271 261 232 225
ball
:, CB a C 0 ,
80 85 76 96 90 81 I, 67
III6
:* cn co YDl CL1 la.2 CD2 c 0 *
96 ee
GLW
k
7
b5
MY
:A ca cc OD1 NO2 c 0 6
1OY
10
GUI 277 :A 270 ca 257 C‘ 264 CD 230 061 225 WE2 223 c 264
Coordinates
10X
2b3
71 61 ii 57 62 72 55 51 53
242 250 255 254 257 252 266 239 22,
26, 265 262 261
3U 32 lb29
243 256 211 237
co
263
17
252
:
241 250
34 60
231 224
0
11 Y CD ::
25, 266 2,1 266 275 272
111 107 120 117 96 93
232 225 232 246 220 209
for
10% 20
109 115 12, 136
102 90 94 90
!A 2
104 114 10, 112
339 137 149 160
63 72 62 69
c 0
120 136
11, 1,)
,e 70
0
22
Y* CB oc C 0
TLR 219 203
35 31
204 205
24
::2 ccl c 0
199 20, 200 196 201
2" 30 43 4, 51
219 22, 226 196 191
:A Cb so C 0
14 TWIl Y 163 CA 177 CB 1n1 cc2 173 OGl 180 c 161 0 155
JB 45 36 23 44 45 36
193 162 170 166 156 163 190
25
15 w CA Cb
155 160 137
55 51 70
17, 178 II)4
CO :i II c 0
127 101 113
:t 71 72 57 b2
::: 200 194 165 155
lb :A
TLlR 112 121
51
lb3
:A CB EG c 0 16 Y :: cn CO c 0
IUX 101 TRP 266 :A 266 CB 254 CO 243 CD1 242 tiE1 233 CL2 226 CD2 234 CL3 231 cz3 221 CO92 216 cz2 219 C 2,7 0 262
us
1:: 136
106 l'o', 102 9,
3”
ELI
SER 130 163 165 115 150 145
140 145 160 166 143 135
:: 93 101 107 116
144 149 130 119 152 lb2
123 111 118 lI7 131 12,
Cb6 169 161 172 159 179 163 TYR
:E CB CG CD1 a1 CI OLY C62 CD2
163 1an 176 174 162 162 173 173 19.6 19b
154 146
lb”
142 153 159 172 178 192 200 213 193 119
c
209 202
153 142
159
161 161 162 162 162 162 160
:: ::: 35 158 311 135 62 154 64 lb5
C66 99 1:: 120 78 73 OR)
,1 a4 9b 95 67 79
145 149 146 145 144 116
'I
9,
146
58 77 54
10, 100 113 11, 911 94
15, 143 150 159 120 121
66
55 b,
290 295
"0 93
224 226
ca %
302 312 287
YO "6 b9
217 225 235
0
295
b"
236
952
along
206 216 223 214 214 204 199 205 201 191 165 IO9 216 205
261 250 250 25a 271 274 263 252 240 239 249 262 262 265
6LR
279 290 296
226 232 244
2b7 276
F ‘
289 265 269
252 236 246
262 290 296
31 ASP N 275 CA 2,0 :t 259
227 229 205 216
294 ,07 309 315
% C 0
2,0 246 2112 292
196 200 222 216
319 317 314 307
CE2 CD2 C 0
264 27, 26, 303 311
209 20, 212 200 206
3b2
%I2 NEYl C 0
243 249 245 306 311
171 173 119 160 169
,I9 317 339 324 322
305 312 3"2 291
169 167 166 192
314 301 299 299
34 N CI C 0
0‘1
,5
Twl
271
365 310 329 321
:A CB
3") 301 3lU
177 179 163
260 266 257
z: :
319 304 292 29,
174 192 151 164
249 266 265
iA
269 261
162 169
259 262
:a2 CC1 CD1 C 0
257 259 243 240 262
166 II4 167 153 164 175
26, 266 256 296 244 14"
h
257 265
155 153
224 23,
::2 CGl CD1
256 265 276 266
154 151 1% 156
211 199 206 219
:
264 244
134 147
226 224
the
3 principal
37
Atomic coordinates are in ?kgstrans (x 10) measured axes a.b and c of the orthorhombic cell (1).
101
:A CB
36
6‘R
:a
b.
29
‘is
13 Y CA
17
107.
53 59 66 65
c
23
CII C‘2 OGl C 0
RESEARCH COMMUNICATIONS
Erabutoxin
10x
O‘Y
:A
CSb
:A cn 60 c 0 h CA Cb co CD1 CL1
Atomic
AND BIOPHYSICAL
:‘E
265
:‘L
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 (continued)
__ k
.__
234
154
223
0
161
230
45 mm. * 138 Ck 13, CB 12* cc.2 121 OGl 131 c 150 0 154 39 k
AR‘
ri
CA C8 CG co
21. 208 215 230 237
133 129 114 111 108
206 193 lb9 191 179
:: Y&k2 NLHl c 0
251 256 269 249 19, 190
106 108 106 113 121 119
162 194 19, 204 196 207
1”
GLP lb6 171 165 1,2
:A c 0
12b 126 129 129
CGl EG2
4, Y CI ce ct
co CE YI c 0
‘LY
43 N CA ce 6G c 0
css
44
PR)
1,l l,, 1.3 151 13, 145 1.9 164 141 132
:o CA ce CG
14, 155 145 153 159
151 164 176 176
146 143 144 I,7
lb9 200 206 215 206 217
141 142 129 129 lS4 152
193 205 212 204 192
165 166 177 19, 160
Molecular given the
1.
structure
respectively. System
Science. aation
procedures
In the
Ramachandran
most residues within
at least
195 185 192 199 178 113
117 114 115 132 101 91
165 155 147 134 139 145
103 93 92 103 98 96
c
55
293 298 301 312 310 293 297
226 232 229 238 240 234 2.5
49 w CI c 0
GLY 229 243 24, 251
293 297 272 267
225 227 225 220
50 X.L 232 * CA 230 ce 219 CG2 214
266 251 246 256
229 22, 235 24s
:",: c 0
243 231 2.9 251
226 21, 212 209
206 20, 22, 215
Atomic
51 :A
were
employed
Cbb 210 199 lb, 179 199 202 css
:A ce SC :
196 19, 209 2o‘J 196 17b
56
‘LO
:*
ce
lb2 170 164
145 140 150
76 69 59
co CG 061 062 c 0
154 160 149 153 173 17,
172 162 18, 170 129 111
:: 60 43 59 46
coordinates
for
angle
(4) and a stereo
plot
are
using
to the
electron
from the
the outer
limit
boundary
The three
drawing
less
principal
Graphics
of Computer
density
restrained
shown
of
Molecular
map and ideali-
mode to be described
apart
are
1 and 2
Department
11,
953
2.5 i structure
the GRIP-75
Carolina,
in an iterative
region.
the
shown in Figs.
obtained
model
(Fig.
54 :A CB SG c 0
of North
were
c
sow 22, 229 2.3 246 2lb 215
cs w
coordinates
results
of this
144 131 130 116 130 119
199 211 120 232 239 252 260 216 211
molecular
22'
210 203 199 199 192 lb5
273 275 26, 269 2',8 2*3 252 291 277
196 lb4 206 204 199 220 21Y
within
lb9 162 161 159 153 167 147 139
192 lb4 177 172 166 161 156 195 206
of the
fall
233 223 221 229 220 239 222 211
27b
2,b 2b3
:o CA C6 ct c 0
plot
LED 225 21b ce 204 CG 193 CO1 lb2 CD2 195 c 211 0 219
271 274
by the University
Fitting
268 263
9m
on the
These
build
177 I76 lb5 195 193 lb2 191
The Ramachandran
based
204 199 IS2 188 X61 167 lb1 lb1 lb9
111 169 159
Conformation.
in Table
244 244 2.7 2.0 263 216 240 232 212
175 179 168 172 154 193 199
lb9
us
236 234 211 218 271 263 296 227 22.
52 :A
157 170 175
ce
4e
42 k Cb c 0
23, 249 251 2.1 265 255 251
us
ce ct CD CL hl c cl
VAL
:A
196 lb6 173 161
51 :,
173
and all molecular
elsewhere. Gly angles,
others lobes
are may
Vol. 88, No. 3, 1979
BIOCHEMICAL
Fig. 3. Erabutoxin double broken lines
be seen
to emerge
curved Cys54
(K49,
2 from
in Fig.
K61)
parallel(3
b. Freehand sketch of alpha-carbon indicate disulfide bridges.
bounded
lobes,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
central
by Cys3 and Cysl7,
are formed
structure
the
by the
(see inset)
backbone
invariant
disulfide
Cys24 and Cys41
five
BADCE strands
as well
as by the
(K*45)r
of the outer
model.
The
core.
These
and by Cys43 and
dominant
anti-
rim segment
43-48
(K49-K55). Fig.
3 shows the molecular
shell-like DC lobe
ring
and 2 residue
neck
region,
molecular
and the underlying tail.
Cys17-Cys24 segments
are
The erabutoxin Apart
from
a very
are no buried
a striking
chemistry.
This
concave
crosslinking molecular
short
(compare
Fig.
invariant
in is
few residues
Nonetheless
chain
a view
"stand"
In the
b molecule
residues
the a structure
in
which
and shows the protruding
concavity,
tral
shape
includes
not
in
toxin
interactions. (facing
but
curved
of the
only
long
cen-
six-membered the
length;
tapered all
other
length.
the
region
of backbone
only
series
is of variable
Because
one peptide the
chain
bonds
numerous
other
in Fig.
stereo-
main-chain
main-chain
interactions, 4) formed
there
to solvent.
molecular
between
thick.
core
inaccessible
of these
the viewer
chain
disulfide
maintains
hydrogen also
only
around
of cross-linking
and elsewhere,
surface
chain
tongue
the
by the C-terminal
open, much of it
or regions degree
curled
formed
4),
emphasizes
atoms and sidethe upper
by the
three
*The sequence enumerations used are that of erabutoxin b and,where different,that of Karlsson (K) which orders all sequence alignments to preserve maximum homology in both short and long chain series.
954
in
Vol. 88, No. 3, 1979
BiOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 4. View (partially schematic) of the erabutoxin the concavity. The reactive site is shown shaded. and Karlsson sequence enumeration used. 0
Invariant position in both toxins only 0 . Conservatively substituted in short chain toxins only Structural residue position.
0 0 lobes
is
inaccessible
particularly the
bonding I
strands
C31*34
(K57)
type
sheet
which
A strand.
for
Backbone.
pattern;
(7),
position 0.
the
chain
in both
molecules
reactive
toxins;
long
from
site
in short
and short
below,
region
chain
chain toxins;
an isolation
(5,6)
shown shaded
in
view.
Polypeptide individual
and short
even to water
significant
projected
long
b molecule looking into One-letter residue code
II.
both
The barreling turn
the
three
(~38)
type
There
involves The general
is Pro11
curve
and twist. 6 bends
III
310 helix) 6 bulge
and Gln12
preference
for
(8)
in strand
complex; hydrogen
are,respectively,A7+BlO and rim
in the
segment
left-hand
B and Gln6
Pro in the
955
is
5 shows the molecular
in the 4 sheet
(i.e.,
a "wide"
Fig.
of the 3 structure
first
47(K53)+E50 edge of the
in the opposite
position
has been
type
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
33
31
T 1 D C Fig. 5. Hydrogen bonding pattern of erabutoxin b polypeptide chain. T;e C=O and H-N groups are joined by dotted lines when the O-N distance 4 3.7 A. Disulfide bridges are shown with striped lines. noted
earlier
(8).
by a "plain and E are strand
Strands
B and C are
+3 connection" joined
leads
into
which
by a right-handed a type
18
bend,
includes
joined
through
a type
2x cross-over 57-60
ring. 956
(K64-K68)
the tapered
I B bend connection
18-21; (9).
in the C-terminal
neck
region
strands
D
The E closed
Vol. 88, No. 3, 1979
BIOCHEMICAL
The dihedral
angles
tain
the B structure,
that
joining
43-54 Gauche. tion
all
Cysl7
(K49-K61)
of erabutoxin Side
Chain
the
series
toxins
In a broad
variant
or type-conserved
. ..32
(K33-K56
shown
shaded
limited
(K45)
are
residues
found
4.
18,19,
or 20 demand conformational
gons
in Fig.
4.
the
which to the
These
chains.
Pro44
stabilizes
bonds
A second probable point
up along
exception functional
play site
four
Va146
(K52)
residues
forms
Glu21
model. region
Most 32-9
of the molecule toxins,
conformational
and
the variations:
found
a key structural
role;
invariant
shown enclosed
glycines,
at positions
in positions
segment
(Gl~/Asp)
turn.
binding
of the main
the
sweeping
part
hollow
of the banked
sorted
"functional"
(5,6).
into
957
With
reactive
approach
of this groupings
of two
residues,
Asn61
(K69)
form
chain. because
one exception,
of the reactive wall
lie
in open hexa-
Two further
and the invariant
segments
they
two in the 6 bends of close
has been designated
have been
substi-
of the
deletions
and are
the rim
in receptor into
(1,2,5,6)
of the short-series minor
in
common in-
series
lobes
of
variations.
of residues
Co-CB
site
two right-hand
to atoms in neighboring
involvement
homologies
features
and long
reactive
invariant)
position
group
Raman solu-
extended
of all
the principal
one or two residue
include series
the type-conserved
laser
and 2 more conservative
may imply
reactive
(one a short
hydrogen
the
evidently
and two
(K50)
bridge
are further
prototype
In the AB loop
neck region
or are bound
are right-handed;
sequence
and distribution
series
by the
tapered
within
main-
are Gauche.Gauche.
of the
positions
define
within
in the
residues
three
The overall
the extent
of common residues
are
which
The disulfide
conclusion
substituted
formed
in Fig.
the
bridges
linkages
left-handed. the other
with
as short
. ..K33)
number
There
is
Three
Distribution.
sense
b structure
disulfide
to 90'.
by 6 more invariants
tutions.
residues
four
b (10).
and conservatively
erabutoxin
of the
close
agree
invariant
of these
lie
and CysQl
findings
Homologous
short
-S-S
is Gauche.Gauche.Trans;
These
studies
about
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
site. concavity. (5,6).
of
all The The Toxicity
Vol. 88, No. 3, 1979
is
less
are
than
found
normal
replaced
hydrophobic direct
BIOCHEMICAL
when some functional in
residues
evidence.
the effect
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
certain have
naturally
consider
chemically
deviant
"functional"
the
findings
are
occurring
been designated
We will
of our recent
residues
toxins.
or
Other
on the basis
structure-function
on the details
modified
of in-
relationships
of the
earlier
and
model
else-
shere. The designations restrictive;
the two
reactive which
"structural"
grouping, binds
roles
for
proper
local
in the
acetylcholine
(K37)
proposed
earlier
is
residue sequence nating
series
continuous
pected
to shift
differences significantly
feature
of invariant
The atomic
Equally,
exclusive.
expression,
as for
and binding
too
In a
may position
some "structural"
association
of the
involves
of identical
somewhat the
the principal
after
homology
the residue residues
may
example,
Gly34
of Asp31
and Arg33
conservative
substitutions
structural
demands
a
alter-
rather
than
a
sequence.
conclusions
refinement. and short
functional
and frequently
or near-identical these
that
stereochemistry
an interrupted
on which
long
neurotoxin
and 8 structure
and/or
coordinates
between
mutually
some instances
(2,5,6,11).
which
segment
are in
residues
functional
defines,
distribution
"functional"
mimetic
a remarkable
distribution
necessarily
to receptor.
(K38)
It
are not
one or more
directly
be required
and "functional"
Model series features
are studies
toxins
based have
do not need
we have described
may be exshown that to affect here
(5,6).
ACKNOWLEDGEMENTS This work was supported by grant NS-07747 from the National Institutes We happily acknowledge the use of GRIP-75 developed by E. G. of Health. Britton, F. P. Brooks, Jr., J. Hermans, J. S. Lipscomb, J. E. McQueen, M. E. Pique, and W. V. Wright REFERENCES 1. 2.
Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko, A. D., and Richardson, J. S. (1976) Proc.Nat. Acad.Sci. U.S. 73, 2991-2994. Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko of the Fifth International A. D., and Richardson, J. S. (1979) Proceedings Symposium Society of Toxinology, Costa Rica (1976), in Toxins - Animal,
958
Vol. 88, No. 3, 1979
3. 4. 5. 6. 7. 8. 9. 10. 11.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Plant and Microbial (P. Rosenberg, ed.) p. 312, Pergamon Press, N.Y. Yang, C. C. (1967) Biochim. Biophys. Acta 133, 346-355. Ramakrishnan, C., and Ramachandran, G. N. (1965) Biophys. J. 5, 909-933. Low, B. W. (1979) in Snake Venoms-Handbook of Experimental Pharmacology (Chen-Yuan Lee, ed.) Vol. 52, pp 213-257, Springer-Verlag, Berlin. Low, B. W. (in press 1979) Proceedings of the Third International Symposium on Cytophannacology in Advances in Cytopharmacology (B. Ceccarelli,F. Clementi, eds.) Vol. 3, pp. 141-147,Raven Press, New York. Venkatachalam, C. M. (1968) Biopolymers 6, 1425-1436. Richardson, J. S.,Getzoff, E. D., and Richardson, D. C. (1978) Proc. Nat. Acad. Sci. U.S. 75, 2574-2578. Richardson, J. S. (1976) Proc. Nat. Acad. Sci. U.S. 73, 2619-2623. Harada, I., Takamatsu, T., Shimanouchi, T., Miyazawa, T., and Tamiya, N. (1976) J. Phys. Chem. 80, 1153-1156. Sato, A. (1977) Ph.D. thesis, Tohoku University, Japan.
959
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
June 13, 1979
Pages 950-959
MOLECULAR CONFORMATION OF ERABUTOXIN b; ATOMIC COORDINATES AT 2.5 i RESOLUTION M. R. Kimball, A. Sate*, Department of Biochemistry,
Received
April
J.
S. Richardson**, L. S. Rosen, and B. W. Lowi Columbia University, 630 W. 168 St.,N.Y.,N.Y.
10032
16,1979
determined from a 2.5 g electron density map are SUMMARY: Atomic coordinates given for erabutoxin b, a sea snake venom postsynaptic neurotoxin. The principal structural features, anti-parallel 8 pleated sheet, 8 bulge and 8 bends are described. The erabutoxin b structure is discussed as structural prototype of this class of homologous curare-mimetic neurotoxins from both land and sea snakes. In this sea snake
communication
we provide
venom neurotoxin,
based
major
features
single
chain
of the molecular protein
of nearly phid)
50 homologous
snake venans
membrane mimetic
distribution
neuromuscular
density
as prototype
from both
to the
erabutoxin
for
Laticauda land
cholinergic
of this
a whole
class
semifasciata,
(elapid) receptor
transmission
b, a
map and describe
and stereochemistry
considered
structural were
features described
of this
of is
and sea
one
(hydro-
at the postsynaptic
in a nondepolarizing
The significance
(1).
was recognized series,
and a reactive short
with
60+62
curare-
disulfide
linkages.
toxin
and a preliminary
of the site
chain
observed
at a
discussion
of the resi-
and conservatively-substituted+ erabutoxin
region
and long
and 7 conservatively-substituted
invariant
short
earlier,
of some common invariant
dues given
iant
w electron
from the sea snake
bind
for
mode.
resolution
toxin
on a 2.5
neurotoxins
which
and block
The main lower
b,
coordinates
conformation
(Mr 6861)
Erabutoxin
neurotoxins.
atomic
described with
71+74
positions These
b structure
latter
as prototype
(1,2).
There
are two
residues,
with
18 invar-
in common, which play
a unique
include
and evident
four struc-
*Present address: Department of Chemistry, Tohoku University, Sendai, Japan. **Permanent address: Department of Anatomy, Duke University, Durham, N.C. t To whom all correspondence should be addressed. +Conservative substitution is used here to define conservation of residue type, i.e., Ser/Thr-hydroxyl group, Glu/Asp-carboxyl group, Val/Leu/Arghydrophobic group and Tyr/His/Trp-aromatic group. 0006-291 Copyright All rights
X/79/1
10950-10$01.00/O
@ 1979 by Academic Press, Inc. of reproduction in anyform reserved.
950
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
-$ Fig. (-1
1. Ramachandran and "outer limit"
Fig. 2. produced
tural they
role; are
Stereo drawing by the GRIP-75
both reduced
native
angle plot for erabutoxin b showing "fully allowed" (----I boundaries. CO1 Gly; [@I Other residues.
of erabutoxin b structure showing Molecular Graphics System.
conformation
and toxicity
(3).
951
are
lost
all
the
residues,
completely
when
Vol. 88, No. 3, 1979
BIOCHEMICAL
TABLE 1
I”& 101 AR‘ L 114 ::, 126 123 1
101
IOX
74 LO 96
93 100 I"2
CL m;
11. 164 165 115 ;::
103 95 100 99 106 92
97 b7 74 65 53 b7
c D
129 122
73 73
112 122
1*1 14Y 154 157 144 143 162 171
61 66 41 39 38 24 65 60
111 123 120 133 112 117 123 116
cc co bb
2
:‘a
L CB CG2 CGl co1 c ‘ 3
lb4 177 174
11 75 96
132 134 133
179
65
152
4
kbb 193 2Ul 216 222 221 22, 234 235 229 1YI 202
62 70 19 66 55 42 41 52
152 163 160 15b 164 161 i49 140 144 176 1,7
cl.
CL2 Ccl2 c 0 5
191 166 17? lb9
159 173 2"O 200
‘A CB ct CD U&l L‘2 c (r
165 198 205 215 212 227 206 220
212 i23 235 24, 254 246 26, 229 234
79
234 22, 233 239 233 242 251 252 247 253
100 90 9, 107 113 121 121 112 102 93
76
70 73 71 66 72 n9 96
203 211 202 20, 199 190 196 219 211
231 236 253 2b1
272 27, 271 261 232 225
ball
:, CB a C 0 ,
80 85 76 96 90 81 I, 67
III6
:* cn co YDl CL1 la.2 CD2 c 0 *
96 ee
GLW
k
7
b5
MY
:A ca cc OD1 NO2 c 0 6
1OY
10
GUI 277 :A 270 ca 257 C‘ 264 CD 230 061 225 WE2 223 c 264
Coordinates
10X
2b3
71 61 ii 57 62 72 55 51 53
242 250 255 254 257 252 266 239 22,
26, 265 262 261
3U 32 lb29
243 256 211 237
co
263
17
252
:
241 250
34 60
231 224
0
11 Y CD ::
25, 266 2,1 266 275 272
111 107 120 117 96 93
232 225 232 246 220 209
for
10% 20
109 115 12, 136
102 90 94 90
!A 2
104 114 10, 112
339 137 149 160
63 72 62 69
c 0
120 136
11, 1,)
,e 70
0
22
Y* CB oc C 0
TLR 219 203
35 31
204 205
24
::2 ccl c 0
199 20, 200 196 201
2" 30 43 4, 51
219 22, 226 196 191
:A Cb so C 0
14 TWIl Y 163 CA 177 CB 1n1 cc2 173 OGl 180 c 161 0 155
JB 45 36 23 44 45 36
193 162 170 166 156 163 190
25
15 w CA Cb
155 160 137
55 51 70
17, 178 II)4
CO :i II c 0
127 101 113
:t 71 72 57 b2
::: 200 194 165 155
lb :A
TLlR 112 121
51
lb3
:A CB EG c 0 16 Y :: cn CO c 0
IUX 101 TRP 266 :A 266 CB 254 CO 243 CD1 242 tiE1 233 CL2 226 CD2 234 CL3 231 cz3 221 CO92 216 cz2 219 C 2,7 0 262
us
1:: 136
106 l'o', 102 9,
3”
ELI
SER 130 163 165 115 150 145
140 145 160 166 143 135
:: 93 101 107 116
144 149 130 119 152 lb2
123 111 118 lI7 131 12,
Cb6 169 161 172 159 179 163 TYR
:E CB CG CD1 a1 CI OLY C62 CD2
163 1an 176 174 162 162 173 173 19.6 19b
154 146
lb”
142 153 159 172 178 192 200 213 193 119
c
209 202
153 142
159
161 161 162 162 162 162 160
:: ::: 35 158 311 135 62 154 64 lb5
C66 99 1:: 120 78 73 OR)
,1 a4 9b 95 67 79
145 149 146 145 144 116
'I
9,
146
58 77 54
10, 100 113 11, 911 94
15, 143 150 159 120 121
66
55 b,
290 295
"0 93
224 226
ca %
302 312 287
YO "6 b9
217 225 235
0
295
b"
236
952
along
206 216 223 214 214 204 199 205 201 191 165 IO9 216 205
261 250 250 25a 271 274 263 252 240 239 249 262 262 265
6LR
279 290 296
226 232 244
2b7 276
F ‘
289 265 269
252 236 246
262 290 296
31 ASP N 275 CA 2,0 :t 259
227 229 205 216
294 ,07 309 315
% C 0
2,0 246 2112 292
196 200 222 216
319 317 314 307
CE2 CD2 C 0
264 27, 26, 303 311
209 20, 212 200 206
3b2
%I2 NEYl C 0
243 249 245 306 311
171 173 119 160 169
,I9 317 339 324 322
305 312 3"2 291
169 167 166 192
314 301 299 299
34 N CI C 0
0‘1
,5
Twl
271
365 310 329 321
:A CB
3") 301 3lU
177 179 163
260 266 257
z: :
319 304 292 29,
174 192 151 164
249 266 265
iA
269 261
162 169
259 262
:a2 CC1 CD1 C 0
257 259 243 240 262
166 II4 167 153 164 175
26, 266 256 296 244 14"
h
257 265
155 153
224 23,
::2 CGl CD1
256 265 276 266
154 151 1% 156
211 199 206 219
:
264 244
134 147
226 224
the
3 principal
37
Atomic coordinates are in ?kgstrans (x 10) measured axes a.b and c of the orthorhombic cell (1).
101
:A CB
36
6‘R
:a
b.
29
‘is
13 Y CA
17
107.
53 59 66 65
c
23
CII C‘2 OGl C 0
RESEARCH COMMUNICATIONS
Erabutoxin
10x
O‘Y
:A
CSb
:A cn 60 c 0 h CA Cb co CD1 CL1
Atomic
AND BIOPHYSICAL
:‘E
265
:‘L
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
TABLE 1 (continued)
__ k
.__
234
154
223
0
161
230
45 mm. * 138 Ck 13, CB 12* cc.2 121 OGl 131 c 150 0 154 39 k
AR‘
ri
CA C8 CG co
21. 208 215 230 237
133 129 114 111 108
206 193 lb9 191 179
:: Y&k2 NLHl c 0
251 256 269 249 19, 190
106 108 106 113 121 119
162 194 19, 204 196 207
1”
GLP lb6 171 165 1,2
:A c 0
12b 126 129 129
CGl EG2
4, Y CI ce ct
co CE YI c 0
‘LY
43 N CA ce 6G c 0
css
44
PR)
1,l l,, 1.3 151 13, 145 1.9 164 141 132
:o CA ce CG
14, 155 145 153 159
151 164 176 176
146 143 144 I,7
lb9 200 206 215 206 217
141 142 129 129 lS4 152
193 205 212 204 192
165 166 177 19, 160
Molecular given the
1.
structure
respectively. System
Science. aation
procedures
In the
Ramachandran
most residues within
at least
195 185 192 199 178 113
117 114 115 132 101 91
165 155 147 134 139 145
103 93 92 103 98 96
c
55
293 298 301 312 310 293 297
226 232 229 238 240 234 2.5
49 w CI c 0
GLY 229 243 24, 251
293 297 272 267
225 227 225 220
50 X.L 232 * CA 230 ce 219 CG2 214
266 251 246 256
229 22, 235 24s
:",: c 0
243 231 2.9 251
226 21, 212 209
206 20, 22, 215
Atomic
51 :A
were
employed
Cbb 210 199 lb, 179 199 202 css
:A ce SC :
196 19, 209 2o‘J 196 17b
56
‘LO
:*
ce
lb2 170 164
145 140 150
76 69 59
co CG 061 062 c 0
154 160 149 153 173 17,
172 162 18, 170 129 111
:: 60 43 59 46
coordinates
for
angle
(4) and a stereo
plot
are
using
to the
electron
from the
the outer
limit
boundary
The three
drawing
less
principal
Graphics
of Computer
density
restrained
shown
of
Molecular
map and ideali-
mode to be described
apart
are
1 and 2
Department
11,
953
2.5 i structure
the GRIP-75
Carolina,
in an iterative
region.
the
shown in Figs.
obtained
model
(Fig.
54 :A CB SG c 0
of North
were
c
sow 22, 229 2.3 246 2lb 215
cs w
coordinates
results
of this
144 131 130 116 130 119
199 211 120 232 239 252 260 216 211
molecular
22'
210 203 199 199 192 lb5
273 275 26, 269 2',8 2*3 252 291 277
196 lb4 206 204 199 220 21Y
within
lb9 162 161 159 153 167 147 139
192 lb4 177 172 166 161 156 195 206
of the
fall
233 223 221 229 220 239 222 211
27b
2,b 2b3
:o CA C6 ct c 0
plot
LED 225 21b ce 204 CG 193 CO1 lb2 CD2 195 c 211 0 219
271 274
by the University
Fitting
268 263
9m
on the
These
build
177 I76 lb5 195 193 lb2 191
The Ramachandran
based
204 199 IS2 188 X61 167 lb1 lb1 lb9
111 169 159
Conformation.
in Table
244 244 2.7 2.0 263 216 240 232 212
175 179 168 172 154 193 199
lb9
us
236 234 211 218 271 263 296 227 22.
52 :A
157 170 175
ce
4e
42 k Cb c 0
23, 249 251 2.1 265 255 251
us
ce ct CD CL hl c cl
VAL
:A
196 lb6 173 161
51 :,
173
and all molecular
elsewhere. Gly angles,
others lobes
are may
Vol. 88, No. 3, 1979
BIOCHEMICAL
Fig. 3. Erabutoxin double broken lines
be seen
to emerge
curved Cys54
(K49,
2 from
in Fig.
K61)
parallel(3
b. Freehand sketch of alpha-carbon indicate disulfide bridges.
bounded
lobes,
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
central
by Cys3 and Cysl7,
are formed
structure
the
by the
(see inset)
backbone
invariant
disulfide
Cys24 and Cys41
five
BADCE strands
as well
as by the
(K*45)r
of the outer
model.
The
core.
These
and by Cys43 and
dominant
anti-
rim segment
43-48
(K49-K55). Fig.
3 shows the molecular
shell-like DC lobe
ring
and 2 residue
neck
region,
molecular
and the underlying tail.
Cys17-Cys24 segments
are
The erabutoxin Apart
from
a very
are no buried
a striking
chemistry.
This
concave
crosslinking molecular
short
(compare
Fig.
invariant
in is
few residues
Nonetheless
chain
a view
"stand"
In the
b molecule
residues
the a structure
in
which
and shows the protruding
concavity,
tral
shape
includes
not
in
toxin
interactions. (facing
but
curved
of the
only
long
cen-
six-membered the
length;
tapered all
other
length.
the
region
of backbone
only
series
is of variable
Because
one peptide the
chain
bonds
numerous
other
in Fig.
stereo-
main-chain
main-chain
interactions, 4) formed
there
to solvent.
molecular
between
thick.
core
inaccessible
of these
the viewer
chain
disulfide
maintains
hydrogen also
only
around
of cross-linking
and elsewhere,
surface
chain
tongue
the
by the C-terminal
open, much of it
or regions degree
curled
formed
4),
emphasizes
atoms and sidethe upper
by the
three
*The sequence enumerations used are that of erabutoxin b and,where different,that of Karlsson (K) which orders all sequence alignments to preserve maximum homology in both short and long chain series.
954
in
Vol. 88, No. 3, 1979
BiOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Fig. 4. View (partially schematic) of the erabutoxin the concavity. The reactive site is shown shaded. and Karlsson sequence enumeration used. 0
Invariant position in both toxins only 0 . Conservatively substituted in short chain toxins only Structural residue position.
0 0 lobes
is
inaccessible
particularly the
bonding I
strands
C31*34
(K57)
type
sheet
which
A strand.
for
Backbone.
pattern;
(7),
position 0.
the
chain
in both
molecules
reactive
toxins;
long
from
site
in short
and short
below,
region
chain
chain toxins;
an isolation
(5,6)
shown shaded
in
view.
Polypeptide individual
and short
even to water
significant
projected
long
b molecule looking into One-letter residue code
II.
both
The barreling turn
the
three
(~38)
type
There
involves The general
is Pro11
curve
and twist. 6 bends
III
310 helix) 6 bulge
and Gln12
preference
for
(8)
in strand
complex; hydrogen
are,respectively,A7+BlO and rim
in the
segment
left-hand
B and Gln6
Pro in the
955
is
5 shows the molecular
in the 4 sheet
(i.e.,
a "wide"
Fig.
of the 3 structure
first
47(K53)+E50 edge of the
in the opposite
position
has been
type
Vol. 88, No. 3, 1979
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
33
31
T 1 D C Fig. 5. Hydrogen bonding pattern of erabutoxin b polypeptide chain. T;e C=O and H-N groups are joined by dotted lines when the O-N distance 4 3.7 A. Disulfide bridges are shown with striped lines. noted
earlier
(8).
by a "plain and E are strand
Strands
B and C are
+3 connection" joined
leads
into
which
by a right-handed a type
18
bend,
includes
joined
through
a type
2x cross-over 57-60
ring. 956
(K64-K68)
the tapered
I B bend connection
18-21; (9).
in the C-terminal
neck
region
strands
D
The E closed
Vol. 88, No. 3, 1979
BIOCHEMICAL
The dihedral
angles
tain
the B structure,
that
joining
43-54 Gauche. tion
all
Cysl7
(K49-K61)
of erabutoxin Side
Chain
the
series
toxins
In a broad
variant
or type-conserved
. ..32
(K33-K56
shown
shaded
limited
(K45)
are
residues
found
4.
18,19,
or 20 demand conformational
gons
in Fig.
4.
the
which to the
These
chains.
Pro44
stabilizes
bonds
A second probable point
up along
exception functional
play site
four
Va146
(K52)
residues
forms
Glu21
model. region
Most 32-9
of the molecule toxins,
conformational
and
the variations:
found
a key structural
role;
invariant
shown enclosed
glycines,
at positions
in positions
segment
(Gl~/Asp)
turn.
binding
of the main
the
sweeping
part
hollow
of the banked
sorted
"functional"
(5,6).
into
957
With
reactive
approach
of this groupings
of two
residues,
Asn61
(K69)
form
chain. because
one exception,
of the reactive wall
lie
in open hexa-
Two further
and the invariant
segments
they
two in the 6 bends of close
has been designated
have been
substi-
of the
deletions
and are
the rim
in receptor into
(1,2,5,6)
of the short-series minor
in
common in-
series
lobes
of
variations.
of residues
Co-CB
site
two right-hand
to atoms in neighboring
involvement
homologies
features
and long
reactive
invariant)
position
group
Raman solu-
extended
of all
the principal
one or two residue
include series
the type-conserved
laser
and 2 more conservative
may imply
reactive
(one a short
hydrogen
the
evidently
and two
(K50)
bridge
are further
prototype
In the AB loop
neck region
or are bound
are right-handed;
sequence
and distribution
series
by the
tapered
within
main-
are Gauche.Gauche.
of the
positions
define
within
in the
residues
three
The overall
the extent
of common residues
are
which
The disulfide
conclusion
substituted
formed
in Fig.
the
bridges
linkages
left-handed. the other
with
as short
. ..K33)
number
There
is
Three
Distribution.
sense
b structure
disulfide
to 90'.
by 6 more invariants
tutions.
residues
four
b (10).
and conservatively
erabutoxin
of the
close
agree
invariant
of these
lie
and CysQl
findings
Homologous
short
-S-S
is Gauche.Gauche.Trans;
These
studies
about
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
site. concavity. (5,6).
of
all The The Toxicity
Vol. 88, No. 3, 1979
is
less
are
than
found
normal
replaced
hydrophobic direct
BIOCHEMICAL
when some functional in
residues
evidence.
the effect
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
certain have
naturally
consider
chemically
deviant
"functional"
the
findings
are
occurring
been designated
We will
of our recent
residues
toxins.
or
Other
on the basis
structure-function
on the details
modified
of in-
relationships
of the
earlier
and
model
else-
shere. The designations restrictive;
the two
reactive which
"structural"
grouping, binds
roles
for
proper
local
in the
acetylcholine
(K37)
proposed
earlier
is
residue sequence nating
series
continuous
pected
to shift
differences significantly
feature
of invariant
The atomic
Equally,
exclusive.
expression,
as for
and binding
too
In a
may position
some "structural"
association
of the
involves
of identical
somewhat the
the principal
after
homology
the residue residues
may
example,
Gly34
of Asp31
and Arg33
conservative
substitutions
structural
demands
a
alter-
rather
than
a
sequence.
conclusions
refinement. and short
functional
and frequently
or near-identical these
that
stereochemistry
an interrupted
on which
long
neurotoxin
and 8 structure
and/or
coordinates
between
mutually
some instances
(2,5,6,11).
which
segment
are in
residues
functional
defines,
distribution
"functional"
mimetic
a remarkable
distribution
necessarily
to receptor.
(K38)
It
are not
one or more
directly
be required
and "functional"
Model series features
are studies
toxins
based have
do not need
we have described
may be exshown that to affect here
(5,6).
ACKNOWLEDGEMENTS This work was supported by grant NS-07747 from the National Institutes We happily acknowledge the use of GRIP-75 developed by E. G. of Health. Britton, F. P. Brooks, Jr., J. Hermans, J. S. Lipscomb, J. E. McQueen, M. E. Pique, and W. V. Wright REFERENCES 1. 2.
Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko, A. D., and Richardson, J. S. (1976) Proc.Nat. Acad.Sci. U.S. 73, 2991-2994. Low, B. W., Preston, H. S., Sato, A., Rosen, L. S., Searl, J. E., Rudko of the Fifth International A. D., and Richardson, J. S. (1979) Proceedings Symposium Society of Toxinology, Costa Rica (1976), in Toxins - Animal,
958
Vol. 88, No. 3, 1979
3. 4. 5. 6. 7. 8. 9. 10. 11.
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
Plant and Microbial (P. Rosenberg, ed.) p. 312, Pergamon Press, N.Y. Yang, C. C. (1967) Biochim. Biophys. Acta 133, 346-355. Ramakrishnan, C., and Ramachandran, G. N. (1965) Biophys. J. 5, 909-933. Low, B. W. (1979) in Snake Venoms-Handbook of Experimental Pharmacology (Chen-Yuan Lee, ed.) Vol. 52, pp 213-257, Springer-Verlag, Berlin. Low, B. W. (in press 1979) Proceedings of the Third International Symposium on Cytophannacology in Advances in Cytopharmacology (B. Ceccarelli,F. Clementi, eds.) Vol. 3, pp. 141-147,Raven Press, New York. Venkatachalam, C. M. (1968) Biopolymers 6, 1425-1436. Richardson, J. S.,Getzoff, E. D., and Richardson, D. C. (1978) Proc. Nat. Acad. Sci. U.S. 75, 2574-2578. Richardson, J. S. (1976) Proc. Nat. Acad. Sci. U.S. 73, 2619-2623. Harada, I., Takamatsu, T., Shimanouchi, T., Miyazawa, T., and Tamiya, N. (1976) J. Phys. Chem. 80, 1153-1156. Sato, A. (1977) Ph.D. thesis, Tohoku University, Japan.
959