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Conformation of the pentasaccharide corresponding to the binding site of heparin to Antithrombin-III
cl
Carbohydrate Research, 165 (1987) cl-c5 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Preliminary communication
Conformation of the pentasaccharide corresponding to the binding site of heparin to Antithrombin-III MASSIMORAGAZZI,DINOR.FERRO, Istituto di Chimica delle Macromolecole
BRUNOPERLY, C.E.A., IRDIIDPC,
91191 Gif-w-Yvette
de1 CNR, via E. Bassini 15, 20133 Milan0 (Italy)
(France)
GIANGIACOMOTORRI,BENITO CASU, Istituto G. Ronzoni,
via G. Colombo 81, 20133 Milan0 (Italy)
PIERRE SINAT, Ecole Normale Suptieure,
Laboratoire
de Chimie, UA 1110, 24 rue Lhomond,
X231 Paris (France)
MAURICEPETITOU,ANDJEAN CHOAY Institut Choay, 46 avenue Th. Gautier, 75016 Paris (France)
(Received January 9th. 1987; accepted for publication, April 16th. 19X7)
The minimal binding site of heparin to Antithrombin-III (AT-III) is represented by a pentasaccharidel-* which was recently synthesised3. We now propose a model for the synthetic pentasaccharide4 1 based on force-field computations and n.m.r. (essentially n.0.e.) measurements. co,-
cli,oso;
CH,OSO,
CH,OSO;
Ho~o~o~o~o~Me NH
OH
:0,A
oso;
NH
I G
NH
I
I
40;
bo3-
1s
AX
AM
A major problem is the determination of the conformation of the iduronate residue I, which has been a matter of controversySc*6. Force-field computations indicated that, in addition to the ‘C, and 4C, conformations, a third almost equienergetic conformation, the skew boat *So, must be considered for the iduronate ring5”. The 3J,,, values for the iduronate residue in various mono- and oligosaccharides related to the heparin binding site for AT-III have been interpreted in 0008-6215/87/$03.50
@ 1987 Elsevier Science Publishers B.V.
c2
PRELIMINARY
0
COM~lII:NI(‘Al‘ION
3% 5
i 0
0
/04 ,’
0
’
0
/
\
0
\
0-s
\
Fig.
I.
The two conformations
short H-Z/H-S
of I, observed in hcpdrin and insitlc
3
2
3
oligosacch:iridc
b
sqocnces.
The
distance in the ?S,, form is indicated.
terms of conformer populationP, the relative abundance of which is governed both by the sequence of monosaccharide units and by the pattern of substitution’h.5d. When present as an internal unit in an oligosaccharidc. the idurtmatc residue is in equilibrium bctwcen the ‘C, and 2S,,conformations (Fig. 1). Force-field computations carried out on 1 provided several models, and the procedure outlined below led to the selection of the most probable. The cp.# energy maps were inspected first for the four pairs of intcrglycosidic torsional angles. Taking into account the three possible conformations of the iduronatc ring, but neglecting differences in the side-chain conformations, sixteen conformers of the pentasacchnridc were found with comparable energy (within 33 kcal.mol-I). Analysis of the J,_, values for 1 indicates no appreciable amount of the 4C, conformation of the iduronate ring St’. , hence the corresponding conformers were discarded. Furthermore. by restricting the cnkrgy range to 2 kcal.mol--I, a scrics of conformers, characterised by the values -7” and 39” for p and I/Iin the A-G intcrglycosidic linkage, can also be discarded. Only the four remaining conformers were then considered; these corrcsponded to the ‘C, and ?S’<, conformations for I,, combined with the fwo possible po.J/ angles for the 1,-A, intcrglycosidic linkage. The values of cp and JI for the four conformers are given in Table I, and the two most stable arc shown in Fig. 2. Using 500-MHz n.m.r. spectroscopy, both intra- and inter-residue negative n.0.e. effects were observed at IO” on a phase-sensitive NOESY map7 (Fig. 3) and quantified by mono-dimensional n.0.e. difference spectroscopy. The n.0.e. observed between H-l and H-2 of the three 2-amino-2-dcoxyglucosc residues was essentially the same (- 16 to - 17%), indicating isotropic tumbling. Particularly noticeable is the strong n.0.e. effect [- 13%. i.e., only slightly smaller than those (17-20%) observed for vicinal diequatorial protons] between H-5 and H-2 of residue I,, as predicted for a prevalent ?So conformation by the above calculations.
PRELIMINARY
c3
COMMUNICATION
b
Fig. 2. The two most stable conformers computed for 1: (a) minimum c I (according sponding to the ‘C, conformation of 1,; (b) minimum s II (conformation %S,).
TABLE
I
INTERGLYCOSIDIC
DIHEDRAL
ANGLES
OF 1, AND THEIR KELATIVE
Minimmu
(cp,; JII)
CI c II SI s II
-36; -36; -36; -36; c
correspond
-26 -26 -26 -27
AND
(p(H-1-GljO-1-C-4)
STABLE CONFORMERS
“Minima
to Table I), corre-
#(C-l-0-lfC-4-H-4)
ENERGIES COMYUTED ACCORDING
--
IN THE FOUR MORE TO REFS. 5a AND
5d
f v2; 45)
CG $3)
(Qe’ tib
E (kcal.mol-‘)
49;4 49;4 50;4 48; 5
-37; -35; -35; -38;
24; -49 31; 19 34; -43 44; 9
0.31 1.34 1.27 0
to the IC, conformation
-33 -31 -41 -44
of Is, whereas
s refers to 2S,.
Indeed, in the skew-boat form, the H-2 to H-5 distance is the shortest interproton distance (2.3 A, as compared with 2.8 A for H-l to H-3), whereas in the chair form it is 4.0 8, (see Fig. 1). This provides support for the occurrence of this conformer. The present picture of the average conformation has the following features. (a) The A-G-A* sequence is quite rigid, owing to a single conformation of the glycosidic linkage. The present calculations indicate that the rigidity of the G-A* linkage arises from the /3-D-glucuronate residue and from the interactions of the peculiar 3-0-sulfo group with O-5 and COO- of G. (b) In the A*-I-A, sequence
PRELIMINARY
COMMUNI(‘ATfON Me
4.80
5.00
Cl,'
i-6
5.20
5.40 /
5.60
/;
5.40
5.2c
5.0 0
4.80
d.60
4.20
4.4 0
4.00
3.50
3.60
+w.m.
3 40
p.p.n.
Fig. 3. %X&MHz phase-sensitive contours have been plotted. {in the four conformers~, mations, and
‘C,
the relative -40%;
NOSY
the iduronate
populations
of AGA”IA,
map (partial)
in :H,O at 20”. Only negative
ring is in equilibrium
computed
from
the presence of the *S, form
between
the s_rm.,values
is confirmed
two confor-
being
‘S,, -60%
by the marked
n.0.e.
effect. Thus,
four
pentasaccharide more
refined
main
conformers
1 in solution force-field
are
proposed
corresponding
and n.0.e.
as possible
to the binding
study is in progress
for
the
site to antithrombin.
candidates
A
to improve
this model.
REFERENCES
1 J. CHOAY, J.-C. LOKMEAU,M. PETITOU,P.
SINAI, AND J. FAREED. Ann. IV. Y. Acud. SC?.. 370 (1981) 644-649. 2 L. THUNBERG,G. BACKSTKOM. AND U. LINDAHL, Curb&y&. Res., 1Of~(1982) 39.FJfO. 3 J. CHOAY, M. Przrirou, J.-C. LORMEAU, P. SINA*. B. CAW. AND G. GArrI. Biachem. ~~~p~y~. Res. C~mmun., 136 (1983) 492499; P. SIWXY.J.-C. JAC‘OWWT. M. P~rrrou, P. DLVHAUSSOY, 1. LEDER. MAN, J. CHOAY. AND G. TOOKRI,Carbohydr. Res., 132 (1984) c:S+9; C. A. A. \A?; BOECKEL, T. BEETZ. J. N. VOS, A. H. M. DF.JO%, S. F. VAN Aeusr, R. H. VANIXN BOSCH.J. M. R. MEHTI:NS.
PRELIMINARY COMMUNICATION
4 5
6 7
C5
AND F. A. VAN DER VLUGY, J. Carbohydr. Chem., 4 (1985) 293-321; Y. ICHIKAWA,R. MONDEN.AND H. KUZUHARA, Tetrahedron Len., (1986) 611-614; M. PETITOU,P. DUCHAUSSOY,I. LEDERMAN,J. CHOAY, P. SINAP, J.-C. JACOUINET,AND G. TORRI, Carbohydr. Res., 147 (1986) 221-236. M. PETITOU,P. DUCHAUSSOY,I. LEDERMAN,J. CHOAY, J.-C. JACQUINET,P. SINAP. AND G. TORRI, Carbohydr. Res., in press. (a) M. RAGAZZI, D. R. FERRO, AND A. PROVASOLI,J. Comput. Chem., 7 (1986) 105-112; (b) G. TORRI, B. CASU, G. GATTITI, M. PETITOU, J. CHOAY, J.-C. JACQUINET,AND P. SINAP, Biochem. Biophys. Res. Commun., 128 (1985) W-140; (c) B. CASU, J. CHOAY, D. R. FERRO, G. GATTI, J.-C. JACQUINET,M. PETIMU, A. PROVASOLI,M. RAGAZZI, P. SINAI, AND G. TORRI, Nature (London), 322 (1986) 215-216; (d) D. R. FERRO, A. PROVASOLI,M. RAGAZZI, G. TORRI, B. CASU, G. GATTI, J.-C. JACQUINET,P. SINAP, M. PETITOU,AND J. CHOAY, J. Am. Chem. Sec., 108 (1986) 6773-6778. D. A. REES, E. R. MORRIS, J. F. STODDART.AND E. S. STEVENS,Nature (London), 317 (1985) 480. G. BODENHAUSEN,H. KOGLER. AND R. R. ERNST,J. Magn. Reson., 58 (1984) 370-388.
Carbohydrate Research, 165 (1987) cl-c5 Elsevier Science Publishers B.V., Amsterdam - Printed in The Netherlands
Preliminary communication
Conformation of the pentasaccharide corresponding to the binding site of heparin to Antithrombin-III MASSIMORAGAZZI,DINOR.FERRO, Istituto di Chimica delle Macromolecole
BRUNOPERLY, C.E.A., IRDIIDPC,
91191 Gif-w-Yvette
de1 CNR, via E. Bassini 15, 20133 Milan0 (Italy)
(France)
GIANGIACOMOTORRI,BENITO CASU, Istituto G. Ronzoni,
via G. Colombo 81, 20133 Milan0 (Italy)
PIERRE SINAT, Ecole Normale Suptieure,
Laboratoire
de Chimie, UA 1110, 24 rue Lhomond,
X231 Paris (France)
MAURICEPETITOU,ANDJEAN CHOAY Institut Choay, 46 avenue Th. Gautier, 75016 Paris (France)
(Received January 9th. 1987; accepted for publication, April 16th. 19X7)
The minimal binding site of heparin to Antithrombin-III (AT-III) is represented by a pentasaccharidel-* which was recently synthesised3. We now propose a model for the synthetic pentasaccharide4 1 based on force-field computations and n.m.r. (essentially n.0.e.) measurements. co,-
cli,oso;
CH,OSO,
CH,OSO;
Ho~o~o~o~o~Me NH
OH
:0,A
oso;
NH
I G
NH
I
I
40;
bo3-
1s
AX
AM
A major problem is the determination of the conformation of the iduronate residue I, which has been a matter of controversySc*6. Force-field computations indicated that, in addition to the ‘C, and 4C, conformations, a third almost equienergetic conformation, the skew boat *So, must be considered for the iduronate ring5”. The 3J,,, values for the iduronate residue in various mono- and oligosaccharides related to the heparin binding site for AT-III have been interpreted in 0008-6215/87/$03.50
@ 1987 Elsevier Science Publishers B.V.
c2
PRELIMINARY
0
COM~lII:NI(‘Al‘ION
3% 5
i 0
0
/04 ,’
0
’
0
/
\
0
\
0-s
\
Fig.
I.
The two conformations
short H-Z/H-S
of I, observed in hcpdrin and insitlc
3
2
3
oligosacch:iridc
b
sqocnces.
The
distance in the ?S,, form is indicated.
terms of conformer populationP, the relative abundance of which is governed both by the sequence of monosaccharide units and by the pattern of substitution’h.5d. When present as an internal unit in an oligosaccharidc. the idurtmatc residue is in equilibrium bctwcen the ‘C, and 2S,,conformations (Fig. 1). Force-field computations carried out on 1 provided several models, and the procedure outlined below led to the selection of the most probable. The cp.# energy maps were inspected first for the four pairs of intcrglycosidic torsional angles. Taking into account the three possible conformations of the iduronatc ring, but neglecting differences in the side-chain conformations, sixteen conformers of the pentasacchnridc were found with comparable energy (within 33 kcal.mol-I). Analysis of the J,_, values for 1 indicates no appreciable amount of the 4C, conformation of the iduronate ring St’. , hence the corresponding conformers were discarded. Furthermore. by restricting the cnkrgy range to 2 kcal.mol--I, a scrics of conformers, characterised by the values -7” and 39” for p and I/Iin the A-G intcrglycosidic linkage, can also be discarded. Only the four remaining conformers were then considered; these corrcsponded to the ‘C, and ?S’<, conformations for I,, combined with the fwo possible po.J/ angles for the 1,-A, intcrglycosidic linkage. The values of cp and JI for the four conformers are given in Table I, and the two most stable arc shown in Fig. 2. Using 500-MHz n.m.r. spectroscopy, both intra- and inter-residue negative n.0.e. effects were observed at IO” on a phase-sensitive NOESY map7 (Fig. 3) and quantified by mono-dimensional n.0.e. difference spectroscopy. The n.0.e. observed between H-l and H-2 of the three 2-amino-2-dcoxyglucosc residues was essentially the same (- 16 to - 17%), indicating isotropic tumbling. Particularly noticeable is the strong n.0.e. effect [- 13%. i.e., only slightly smaller than those (17-20%) observed for vicinal diequatorial protons] between H-5 and H-2 of residue I,, as predicted for a prevalent ?So conformation by the above calculations.
PRELIMINARY
c3
COMMUNICATION
b
Fig. 2. The two most stable conformers computed for 1: (a) minimum c I (according sponding to the ‘C, conformation of 1,; (b) minimum s II (conformation %S,).
TABLE
I
INTERGLYCOSIDIC
DIHEDRAL
ANGLES
OF 1, AND THEIR KELATIVE
Minimmu
(cp,; JII)
CI c II SI s II
-36; -36; -36; -36; c
correspond
-26 -26 -26 -27
AND
(p(H-1-GljO-1-C-4)
STABLE CONFORMERS
“Minima
to Table I), corre-
#(C-l-0-lfC-4-H-4)
ENERGIES COMYUTED ACCORDING
--
IN THE FOUR MORE TO REFS. 5a AND
5d
f v2; 45)
CG $3)
(Qe’ tib
E (kcal.mol-‘)
49;4 49;4 50;4 48; 5
-37; -35; -35; -38;
24; -49 31; 19 34; -43 44; 9
0.31 1.34 1.27 0
to the IC, conformation
-33 -31 -41 -44
of Is, whereas
s refers to 2S,.
Indeed, in the skew-boat form, the H-2 to H-5 distance is the shortest interproton distance (2.3 A, as compared with 2.8 A for H-l to H-3), whereas in the chair form it is 4.0 8, (see Fig. 1). This provides support for the occurrence of this conformer. The present picture of the average conformation has the following features. (a) The A-G-A* sequence is quite rigid, owing to a single conformation of the glycosidic linkage. The present calculations indicate that the rigidity of the G-A* linkage arises from the /3-D-glucuronate residue and from the interactions of the peculiar 3-0-sulfo group with O-5 and COO- of G. (b) In the A*-I-A, sequence
PRELIMINARY
COMMUNI(‘ATfON Me
4.80
5.00
Cl,'
i-6
5.20
5.40 /
5.60
/;
5.40
5.2c
5.0 0
4.80
d.60
4.20
4.4 0
4.00
3.50
3.60
+w.m.
3 40
p.p.n.
Fig. 3. %X&MHz phase-sensitive contours have been plotted. {in the four conformers~, mations, and
‘C,
the relative -40%;
NOSY
the iduronate
populations
of AGA”IA,
map (partial)
in :H,O at 20”. Only negative
ring is in equilibrium
computed
from
the presence of the *S, form
between
the s_rm.,values
is confirmed
two confor-
being
‘S,, -60%
by the marked
n.0.e.
effect. Thus,
four
pentasaccharide more
refined
main
conformers
1 in solution force-field
are
proposed
corresponding
and n.0.e.
as possible
to the binding
study is in progress
for
the
site to antithrombin.
candidates
A
to improve
this model.
REFERENCES
1 J. CHOAY, J.-C. LOKMEAU,M. PETITOU,P.
SINAI, AND J. FAREED. Ann. IV. Y. Acud. SC?.. 370 (1981) 644-649. 2 L. THUNBERG,G. BACKSTKOM. AND U. LINDAHL, Curb&y&. Res., 1Of~(1982) 39.FJfO. 3 J. CHOAY, M. Przrirou, J.-C. LORMEAU, P. SINA*. B. CAW. AND G. GArrI. Biachem. ~~~p~y~. Res. C~mmun., 136 (1983) 492499; P. SIWXY.J.-C. JAC‘OWWT. M. P~rrrou, P. DLVHAUSSOY, 1. LEDER. MAN, J. CHOAY. AND G. TOOKRI,Carbohydr. Res., 132 (1984) c:S+9; C. A. A. \A?; BOECKEL, T. BEETZ. J. N. VOS, A. H. M. DF.JO%, S. F. VAN Aeusr, R. H. VANIXN BOSCH.J. M. R. MEHTI:NS.
PRELIMINARY COMMUNICATION
4 5
6 7
C5
AND F. A. VAN DER VLUGY, J. Carbohydr. Chem., 4 (1985) 293-321; Y. ICHIKAWA,R. MONDEN.AND H. KUZUHARA, Tetrahedron Len., (1986) 611-614; M. PETITOU,P. DUCHAUSSOY,I. LEDERMAN,J. CHOAY, P. SINAP, J.-C. JACOUINET,AND G. TORRI, Carbohydr. Res., 147 (1986) 221-236. M. PETITOU,P. DUCHAUSSOY,I. LEDERMAN,J. CHOAY, J.-C. JACQUINET,P. SINAP. AND G. TORRI, Carbohydr. Res., in press. (a) M. RAGAZZI, D. R. FERRO, AND A. PROVASOLI,J. Comput. Chem., 7 (1986) 105-112; (b) G. TORRI, B. CASU, G. GATTITI, M. PETITOU, J. CHOAY, J.-C. JACQUINET,AND P. SINAP, Biochem. Biophys. Res. Commun., 128 (1985) W-140; (c) B. CASU, J. CHOAY, D. R. FERRO, G. GATTI, J.-C. JACQUINET,M. PETIMU, A. PROVASOLI,M. RAGAZZI, P. SINAI, AND G. TORRI, Nature (London), 322 (1986) 215-216; (d) D. R. FERRO, A. PROVASOLI,M. RAGAZZI, G. TORRI, B. CASU, G. GATTI, J.-C. JACQUINET,P. SINAP, M. PETITOU,AND J. CHOAY, J. Am. Chem. Sec., 108 (1986) 6773-6778. D. A. REES, E. R. MORRIS, J. F. STODDART.AND E. S. STEVENS,Nature (London), 317 (1985) 480. G. BODENHAUSEN,H. KOGLER. AND R. R. ERNST,J. Magn. Reson., 58 (1984) 370-388.