BIOCHEMICAL
Vol. 64, No. 1,1975
IZ!WVLRICI~~AiiD DIVmE
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
CATIONS: CRYSTALSTRUCTURC OF i'hE BARIUMCOIG'LEX
Jean A. hamilton,
L. K. Steinrauf
and 6radford Braden
Departments of Biochemistry and biophysics Indiana University School of Medicine Indianapolis, Indiana 40202
Received
March
4,1975
The molecular structure of the 2:2 complex of the cyclohexadepsipeptide antibiotic beauvericin with barium picrate has been determined by X-ray crystallography. The structure serves to confirm previous observations on the bimolecular bellavior of beauvericin and of the ions transrorted by beauverh-l. The intimate involvement of the anions in the coordination of the barium also explains observations that the cation specificity of beauvericin in membranetransport depends on the species of anions present.
beauvericin phenylalanyl
(1) is a cyclic
and L-cc hydrov)
membranetransport mitochondria (2,3). effectiveness
hexadepsipentide of alternating isovaleryl
D-G&Methyl)-
residues known to be active
of monovalent metal cations in biological
in the
systems such as
In previous corrununications we have established also the
of beauvericin with group 11 A cations in solvent extraction
and in U-tube transport
(4) for which Da > Ca, and in liposomes and bacterial
chromatophores (5) for which Ca > Ba. This crystal was undertaken in an effort
structure
determination
to explain these observations. ME?HODS
The beauvericin was synthesized in the laboratory and is the sameas used previously
(4).
of Dr. Roger Koeske
Crystals gown from a mixture of
chloroform and toluene were onthorhombic, space group P2l2l2 with a = 28.05(g), b= 15.79(4),
c = 16.99(6) i.
This is one of five
bv Ea Pic2, and is called form B. bv - barium picrate structure. Copyright All rights
Three
crystal
of the four other crystal
are obviously alternative
151
forms of
packing forms of the present
Only form E shows no obvious relationship.
Q 1975 b.v Academic Press. Inc. of reproductiorl irl a,zy fh~f resetwd.
forms (4) of
Vol. 64, No. 1,1975
BfOCHEMlCAL
Data were collected resolution,
AND BIOPHYSICAL RESEARCH COMMJNiCATlONS
on the Supper-Pace automatic diffractometer
giving 2,500 non-zero independent reflections,
the barium atoms were readily cycles of structure the entire
factor
structure.
Ihe positions of
obtained from a Patterson synthesis.
and Fourier calculations
Calculations
to 1.1 i
Several
enabled us to recognize
were madeusing Stewart's X-ray 72 system
of programs, CDC-6600version. RESULTS The molecular structure structure
The
has as its center two barium ions separated by the very close dis-
tance of 4.13 i. picrate
thus found was (Bv*Ea.Pic3.Ba.Bv)'Pic-.
These two cations are bridged and held together by three
anions extending outward radially
each picrate
contributing
like a three-bladed propeller
two oxygen atoms to the coordination
of each barium.
The two beauvericin molecules are located at each end of the propeller Each beauvericin isovaleryl
contributes
residues.
shaft.
the three car-bony1 oxygen atoms from the three a-
The benzene ring of the phenylalanyl
the packing to enclose the anions. three-fold
with
residues provides
The complex has a non-crystallographic
axis of symmetry about the line connecting the barium atams. The
fourth picrate
ion is found somedistance away along with two toluene nu3le-
cules, none of which are well resolved.
Figure 1 is a drawing from the side
of the complex, and Figure 2 is a view down the non-crystallographic
three-
fold axis. DISCUSSION Picrate ions were not included in any of the many studies of the behavior of beauvericin
in membranesystems.
portant part in the structure
Since the picrate
we have presented here, it
ions play such an imis necessary either
to nominate one or more other anions or to propose a new or at least modified complex for membranetransport.
Looking at our present structure
it would
seempossible that the complex (Bv.M+.Bv) might be stable for monovalent cations.
However, the six-fold
coordination
thus provided would probably be in-
adequate for barium and possibly calcium ions as well.
152
BIOCHEMICAL
Vol. 64, No. 1,1975
Fig. 1.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The (Bv*Ba*Pic3*Ba*Bv)+ complex as seen from the side. picrate and the two solvent molecules are not shown.
Fig. 2.
Only part of the distant
The samecomplex viewed down the end. beauvericin is given.
In order to preserve use of the present structure substitute
other anions for picrate.
vious choice. nearly the
same
as
that
found
(2) found that,beauvericin
ificities
it might be possible to
acids would be the most ob-
Indeed, the oxygen to oxygen distance in carboxylates
mmbrane studies of beauvericin &.
Carboxylic
The isbound
with different
in
Three separate observations from
picrate.
support this suggestion.
First,
in mitochondria showed different
carboxylic
is very
acids
153
present.
Estrada-O.etcation spec-
It is easy to see hm the
Vol. 64, No. 1,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
change of anion would probably change the cation specificity structure.
Second, Prince,
Crofts,
and Steinrauf
(5) found an apparent
charge of plus one for calcium in the membranetransprt matophores in the presence of beauvericin. of anions with the calcium.
transport
dependence on beauvericin
in lipid
(6) for both monoand divalent nolecules per transporting
cations,
ing ability
membraneshas been observed by us
thus preserving the two beauvericin
unit.
in place of phenylalanine, of beauvericin
rings of the phenylalanyl
(5).
which has valine,
does not have the divalent
The answer must certainly
leucine,
complex and thus to achieve a lower net charge.
of complexes with polyvalent
the phenylalanyl
the benzene
to incorporate
The charge per surface area is probably an important consideration
the beauvericin
or
cation bind-
involve
residues which allow beauvericin
anions into the transporting
stability
chro-
a second-order concentration
It may now be nossible to ask why enniatin, isoleucine
of bacterial
This could be accounted for by the
Third,
bilayer
of the present
cations.
In addition
in the
to binding anions
complex reduces charge per surface area by the large area of residues.
The present structure
also suggests that the N-methyl groups could well
be replaced by larger groups such as isopropyl without changing the activity toward divalent
cations.
The site of the potassium ion of the crystal was postulated
structure
of enniatin
* KI
(7) to be inside the cage of the six carbonyl oxygen atoms.
In view of our present results this is probably incorrect
since it would place
the potassium too close to the carbonyl carbon atoms. Whenthe refinement of our present structure
is complete we plan to undertake calculations
binding energy of the cation in alternative
for the
positions and also for the bind-
ing of other anions. The present structure
represents quite a different
than that detemined by us (8) previously
for the dodecapeptide valinomycin
which a potassium ion was found to be entirely
154
way of binding cations
enclosed by the polypeptide
in
BIOCHEMICAL
Vol. 64, No. 1,1975
chain and not near the anion. resulted
in any influence
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
None of the rrany studies on valinomycin has
of the anion on the cation specificity.
to change the cation specificity
it would presumably be necessary to alter
the amino acid sequence. Thus beauvericin, ion specificity
altered
after
In order
with the ability
to have its cat-
synthesis by the binding of different
anions,
represents a n-ore advanced concept of function. In many ways beauvericin valinomycin and alamethicin both cation and antibiotic
nay be considered to be an intermediate (9).
between
Because of the second-order dependence on
(6) beauvericin will
go from negligible
to full
saturation
my&l.
Such behavior is somewhatsimilar to the off-on behavior of alamethicin
(9).
in a much smaller range of concentration
transport
Also, beauvericin,
which is first-order
than would valino-
second-order, is intermediate between valinomycin,
with respect to cation concentration,
and the sixth-order
behavior of alamethicin.
X-RAY 72 system of crystallographic
computer programs is the creation
L&. James Stewart, Computer Science Service, beauvericin
University
of Maryland.
was synthesized by Mr. Sherwin Isaac in the laboratory
Roger Roeske. Financial
of
The
of Dr.
support has been from National Science Foundation
Grant GB 24192. Cl%6600 computer time was furnished by the Research Computing Service,
Indiana University-Purdue
crystallography
facilities
University
at Indianapolis,
and the
were supported by the Heart Research Center, Grant
HE 06307 from the National Heart Institute. REFERENCES 1.
Hamill, R. L., Higgins, C. E., Boaz, H. E., and Gorman, M. (1969) Tetrahedron Letters 4255-4258.
2.
Estrada-0,
S., Gomez-Louer-o, C., and Montal, M. (1972) Bioenergetics 3,
417-428. 3.
I!orschner, E., and Lardy, H. (1968) Antimicrobial pp. 11-14.
4.
Roeske, R. W., Isaac, S., king, T. E., and Steinrauf, them. Biophys. Res. Comunun. 57, 554-561.
155
Agents and Chemotherapy L. K. (1974) Bio-
Vol. 64, No. 1,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5.
Prince, R. C., Crofts, A. R., and Steinrauf, phys. Res. Cormun. 59, 697-703.
6.
Yafuso , M. , Freeman, A. R., Steinrauf, Fed. Pmt. Abstracts 33, 1258.
7.
Dobler, M., Dunitz, J. D., and Krajewski, J. (1969) J. Mol. Biol. E, 603-606.
8.
Pinkerton, M., Steinrauf, L. K., and Dawkins, P. (1969) Biochem. Biophys. Res. Cmun. 35, 512-518.
9.
Cherry, R. J. (1972) Chem. Phys. Lipids, S, 393-400; Gordon, L. G. M., and Haydon, D. A. (1972) Biochim. Bionhys. Acta 255, 1014-1018.
156
L. K. (1974) Eiochem. Bio-
L. K., and Roeske, R. W. (1974)
Vol. 64, No. 1,1975
IZ!WVLRICI~~AiiD DIVmE
AND BIOPHYSICAL
RESEARCH COMMUNICATIONS
CATIONS: CRYSTALSTRUCTURC OF i'hE BARIUMCOIG'LEX
Jean A. hamilton,
L. K. Steinrauf
and 6radford Braden
Departments of Biochemistry and biophysics Indiana University School of Medicine Indianapolis, Indiana 40202
Received
March
4,1975
The molecular structure of the 2:2 complex of the cyclohexadepsipeptide antibiotic beauvericin with barium picrate has been determined by X-ray crystallography. The structure serves to confirm previous observations on the bimolecular bellavior of beauvericin and of the ions transrorted by beauverh-l. The intimate involvement of the anions in the coordination of the barium also explains observations that the cation specificity of beauvericin in membranetransport depends on the species of anions present.
beauvericin phenylalanyl
(1) is a cyclic
and L-cc hydrov)
membranetransport mitochondria (2,3). effectiveness
hexadepsipentide of alternating isovaleryl
D-G&Methyl)-
residues known to be active
of monovalent metal cations in biological
in the
systems such as
In previous corrununications we have established also the
of beauvericin with group 11 A cations in solvent extraction
and in U-tube transport
(4) for which Da > Ca, and in liposomes and bacterial
chromatophores (5) for which Ca > Ba. This crystal was undertaken in an effort
structure
determination
to explain these observations. ME?HODS
The beauvericin was synthesized in the laboratory and is the sameas used previously
(4).
of Dr. Roger Koeske
Crystals gown from a mixture of
chloroform and toluene were onthorhombic, space group P2l2l2 with a = 28.05(g), b= 15.79(4),
c = 16.99(6) i.
This is one of five
bv Ea Pic2, and is called form B. bv - barium picrate structure. Copyright All rights
Three
crystal
of the four other crystal
are obviously alternative
151
forms of
packing forms of the present
Only form E shows no obvious relationship.
Q 1975 b.v Academic Press. Inc. of reproductiorl irl a,zy fh~f resetwd.
forms (4) of
Vol. 64, No. 1,1975
BfOCHEMlCAL
Data were collected resolution,
AND BIOPHYSICAL RESEARCH COMMJNiCATlONS
on the Supper-Pace automatic diffractometer
giving 2,500 non-zero independent reflections,
the barium atoms were readily cycles of structure the entire
factor
structure.
Ihe positions of
obtained from a Patterson synthesis.
and Fourier calculations
Calculations
to 1.1 i
Several
enabled us to recognize
were madeusing Stewart's X-ray 72 system
of programs, CDC-6600version. RESULTS The molecular structure structure
The
has as its center two barium ions separated by the very close dis-
tance of 4.13 i. picrate
thus found was (Bv*Ea.Pic3.Ba.Bv)'Pic-.
These two cations are bridged and held together by three
anions extending outward radially
each picrate
contributing
like a three-bladed propeller
two oxygen atoms to the coordination
of each barium.
The two beauvericin molecules are located at each end of the propeller Each beauvericin isovaleryl
contributes
residues.
shaft.
the three car-bony1 oxygen atoms from the three a-
The benzene ring of the phenylalanyl
the packing to enclose the anions. three-fold
with
residues provides
The complex has a non-crystallographic
axis of symmetry about the line connecting the barium atams. The
fourth picrate
ion is found somedistance away along with two toluene nu3le-
cules, none of which are well resolved.
Figure 1 is a drawing from the side
of the complex, and Figure 2 is a view down the non-crystallographic
three-
fold axis. DISCUSSION Picrate ions were not included in any of the many studies of the behavior of beauvericin
in membranesystems.
portant part in the structure
Since the picrate
we have presented here, it
ions play such an imis necessary either
to nominate one or more other anions or to propose a new or at least modified complex for membranetransport.
Looking at our present structure
it would
seempossible that the complex (Bv.M+.Bv) might be stable for monovalent cations.
However, the six-fold
coordination
thus provided would probably be in-
adequate for barium and possibly calcium ions as well.
152
BIOCHEMICAL
Vol. 64, No. 1,1975
Fig. 1.
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
The (Bv*Ba*Pic3*Ba*Bv)+ complex as seen from the side. picrate and the two solvent molecules are not shown.
Fig. 2.
Only part of the distant
The samecomplex viewed down the end. beauvericin is given.
In order to preserve use of the present structure substitute
other anions for picrate.
vious choice. nearly the
same
as
that
found
(2) found that,beauvericin
ificities
it might be possible to
acids would be the most ob-
Indeed, the oxygen to oxygen distance in carboxylates
mmbrane studies of beauvericin &.
Carboxylic
The isbound
with different
in
Three separate observations from
picrate.
support this suggestion.
First,
in mitochondria showed different
carboxylic
is very
acids
153
present.
Estrada-O.etcation spec-
It is easy to see hm the
Vol. 64, No. 1,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
change of anion would probably change the cation specificity structure.
Second, Prince,
Crofts,
and Steinrauf
(5) found an apparent
charge of plus one for calcium in the membranetransprt matophores in the presence of beauvericin. of anions with the calcium.
transport
dependence on beauvericin
in lipid
(6) for both monoand divalent nolecules per transporting
cations,
ing ability
membraneshas been observed by us
thus preserving the two beauvericin
unit.
in place of phenylalanine, of beauvericin
rings of the phenylalanyl
(5).
which has valine,
does not have the divalent
The answer must certainly
leucine,
complex and thus to achieve a lower net charge.
of complexes with polyvalent
the phenylalanyl
the benzene
to incorporate
The charge per surface area is probably an important consideration
the beauvericin
or
cation bind-
involve
residues which allow beauvericin
anions into the transporting
stability
chro-
a second-order concentration
It may now be nossible to ask why enniatin, isoleucine
of bacterial
This could be accounted for by the
Third,
bilayer
of the present
cations.
In addition
in the
to binding anions
complex reduces charge per surface area by the large area of residues.
The present structure
also suggests that the N-methyl groups could well
be replaced by larger groups such as isopropyl without changing the activity toward divalent
cations.
The site of the potassium ion of the crystal was postulated
structure
of enniatin
* KI
(7) to be inside the cage of the six carbonyl oxygen atoms.
In view of our present results this is probably incorrect
since it would place
the potassium too close to the carbonyl carbon atoms. Whenthe refinement of our present structure
is complete we plan to undertake calculations
binding energy of the cation in alternative
for the
positions and also for the bind-
ing of other anions. The present structure
represents quite a different
than that detemined by us (8) previously
for the dodecapeptide valinomycin
which a potassium ion was found to be entirely
154
way of binding cations
enclosed by the polypeptide
in
BIOCHEMICAL
Vol. 64, No. 1,1975
chain and not near the anion. resulted
in any influence
AND BIOPHYSICAL RESEARCH COMMUNlCATlONS
None of the rrany studies on valinomycin has
of the anion on the cation specificity.
to change the cation specificity
it would presumably be necessary to alter
the amino acid sequence. Thus beauvericin, ion specificity
altered
after
In order
with the ability
to have its cat-
synthesis by the binding of different
anions,
represents a n-ore advanced concept of function. In many ways beauvericin valinomycin and alamethicin both cation and antibiotic
nay be considered to be an intermediate (9).
between
Because of the second-order dependence on
(6) beauvericin will
go from negligible
to full
saturation
my&l.
Such behavior is somewhatsimilar to the off-on behavior of alamethicin
(9).
in a much smaller range of concentration
transport
Also, beauvericin,
which is first-order
than would valino-
second-order, is intermediate between valinomycin,
with respect to cation concentration,
and the sixth-order
behavior of alamethicin.
X-RAY 72 system of crystallographic
computer programs is the creation
L&. James Stewart, Computer Science Service, beauvericin
University
of Maryland.
was synthesized by Mr. Sherwin Isaac in the laboratory
Roger Roeske. Financial
of
The
of Dr.
support has been from National Science Foundation
Grant GB 24192. Cl%6600 computer time was furnished by the Research Computing Service,
Indiana University-Purdue
crystallography
facilities
University
at Indianapolis,
and the
were supported by the Heart Research Center, Grant
HE 06307 from the National Heart Institute. REFERENCES 1.
Hamill, R. L., Higgins, C. E., Boaz, H. E., and Gorman, M. (1969) Tetrahedron Letters 4255-4258.
2.
Estrada-0,
S., Gomez-Louer-o, C., and Montal, M. (1972) Bioenergetics 3,
417-428. 3.
I!orschner, E., and Lardy, H. (1968) Antimicrobial pp. 11-14.
4.
Roeske, R. W., Isaac, S., king, T. E., and Steinrauf, them. Biophys. Res. Comunun. 57, 554-561.
155
Agents and Chemotherapy L. K. (1974) Bio-
Vol. 64, No. 1,1975
BIOCHEMICAL
AND BIOPHYSICAL RESEARCH COMMUNICATIONS
5.
Prince, R. C., Crofts, A. R., and Steinrauf, phys. Res. Cormun. 59, 697-703.
6.
Yafuso , M. , Freeman, A. R., Steinrauf, Fed. Pmt. Abstracts 33, 1258.
7.
Dobler, M., Dunitz, J. D., and Krajewski, J. (1969) J. Mol. Biol. E, 603-606.
8.
Pinkerton, M., Steinrauf, L. K., and Dawkins, P. (1969) Biochem. Biophys. Res. Cmun. 35, 512-518.
9.
Cherry, R. J. (1972) Chem. Phys. Lipids, S, 393-400; Gordon, L. G. M., and Haydon, D. A. (1972) Biochim. Bionhys. Acta 255, 1014-1018.
156
L. K. (1974) Eiochem. Bio-
L. K., and Roeske, R. W. (1974)
