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Determination of the Bioactive Conformation of the Carbohydrate Ligand in the E-SelectinSialyl LewisX Complex.

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Determination of the Bioactive Conformation
of the Carbohydrate Ligand in the E-Selectin/
Sialyl LewisYComplex**
Karoline Scheffler. Beat Ernst, Andreas Katopodis,
John L M,ignani, Wey Tong Wang,
Rudiger Wciceinann. and Thomas Peters*
The expression of E-selectin receptors on the surface of
endothelial cells stimulated by cytokines i s one of the first steps
in inflammatory processes.[" E-selectin i s a membrane-resident ylycoprotein belonging to the selectin family (E-selectin.
L-selectin, P-selectin) which specifically binds the sialyl Lewis"
antigen present on neutrophilic granulocytes. To understand the
recopnition :ind binding processes at the inolecular level. the
tliree-dimcnsionai ( 3 D ) structure of the complex formed between E-selectin and sialyl Lewis' must be analyzed. In this
connection ;I considerable advance was marked by the elucidat a l structure of E-selectin,[3]Unequivocal exper)r the determination of the conformation of the
ligand sialyl Lewis' in the E-selectin complex have been lacking
until now. Although two recent publications['. 'I described
iransfer NOE: experiments['' for the conformational analysis of
coinplexcd sialyl Lewis'. diffeient conformations for the bound
ligand wcre given und the orientation of the fucose residue in the
complex wa\ not even mentioned.
We now describe transfer NOE experiments on the E-selectin;
complex. The tetrasaccharide sialyl Lewis" was
thetically;[71the E-selectin used is a recombinant
-selectin and human IgG. in which the lectin. the
EGF. and sir; ('R domains of the E-selectin replace the antigen
binding sites i i i IgG.['] NOESY spectra were recorded with difrerent mixing times to determine the transfer NOES.['' In order
to suppress the interfering proton resonance >ipnalsof the E-selectin and thus enable the quantitative evaluation of the spectra.
a spinlock filterrio1was used. For comparison purposes NOESY
experiments were performed on pure E-selectin and free sialyl
Lewisx.['I ' The assignments of the proton rcsonance signals
were carried out using available data.["'
Comparison of the NOESY spectra of E-wlectin and free
sialyl Lewis" shows that no interfering superposition of proton
signals from the glycan portion of E-selectin occurs with those
of the tetrasaccharide. As reported in previous publications.["'
sinall negative NOEs are observed for free sialyl Lewis"
(Fig. 1 a). Interglycosidic NOEs occur which correspond with
known NOEs; however. no interglycosidic NOE i s observed
Fig. 1 . 1D NOESY spectra 19.1 I]of l'ree sialyl Lewis' f ; i ) :ind sialyl L e ~ i s com'
pleied with E-selectin ( h i . Spectrum it wiis recorded w,ith ii iniiuing time of 900 ms
without a spinlock filter. spectrum b with ii mixing time of I T 0 ins and ;I spinlock
Table 1. 'H N M R chemical shifts ( 8 values) for the tetrasaccharide sialyl Lewis' in
between H8Nand H3" as was found in ROESY experiment^."^]
D,O at 303 K and 600 MHL. Reference signal: 6(HDO) = 4.722.
The corresponding effect was also observed with sialyLa(2 -+ 3)N-acetyllactosaniine.[12a1
The reason for these differing results is
NeuNAc Gal
GlcNAc Fuc
that the correlation time T~ of the tetrasaccharide leads to a
value 0 ~ (w
7 ~= spectrometer frequency, 600 MHz) of approxiHI
mately 1. In this region the intensities of the NOEs are extremely
4 x C H , 1.28
sensitive to even small changes in the local mobility of individual
3 82
1 x C H , 1.52
parts of the molecule. This is clearly shown in the C9 chain at the
reducing end of sialyl Lewis" for which no, or even very weak
positive, NOEs are observed. Similarly the glycerol side chain of
the N-acetylneuraminic acid residue may show a local correla3.88
tion time T that deviates from the global correlation time t,,
such that NOEs in this side chain, for example NOE H8N/H3G,
3 64
are no longer detectable. Taking into consideration the experi3.87
ments carried out by other research groups["" l 3 I it appears
that, in general, the sialyl Lewis" tetrasaccharide exists in solution as an equilibrium of several conformation^,['^] which to a
considerably from the corresponding NOEs in free sialyl Lewis"
large extent are characterized by the orientation of the N-acetylneuraminic acid residue. The 'H N M R data for the tetrasaccha(Figs. 1 and 2). The most obvious difference is the absence of a
transfer NOE between H3rx and H3'. However, a clear transfer
ride are summarized in Table 1 .
NOE between HfJN and H3G is observed (Figs. 2 b and 2c). A
Even with a mixing time of 25 ms, transfer NOEs occur for
the complex. Since complete transfer NOE curves were obtained
weak transfer NOE is observed between the methyl group of the
N-acetyl function attached to C5Nand H7Nwithin the N-acetylby recording- NOESY suectra at different mixing times,"' spin
diffusion effects can be excluded from the following
discussion of the NOEs.
The intensities of the trans'
fer NOEs and the position
of the maxima in the transH5G
fer NOE curves clearly indicate that the observed effects are indeed transfer
NOEs. Transfer NOEs between E-selectin and sialyl
Lewis" protons could not
be observed at the selected
protein:ligand ratio."] The
following discussion concerns only the NOESY
spectrum recorded with a
mixing time of 150ms
(Fig. 1 b). A precise analysis of the transfer NOE data
and other N M R experiments in particular with
consideration of exchange
equilibria and the involvement of the amino acid prot o n ~ " ~ in
] the binding
pocket of E-selectin will be
published elsewhere. For
comparison the corresponding section from the
2D NOESY spectrum of
free sialyl Lewis" is shown
in Figure l a . Sections of
the corresponding
NOESY spectra relevant to
the discussion of the most
important effects are col5
lected in Figure 2.
The interglycosidic transFig 2 . 1D representations of the NOESY spectra from Figure 1, obtained by the addition of traces of F1. The lower traces are taken
fer NOEs between the Nfrom the NOESY spectrum of free sialyl Lewis' (cf. Fig. 1a ) ; the upper traces correspond to complexed sialyl Lewisx (cf. Fig. 1 h).
acetylneuraminic acid and
a) 6 = H l ' : b) 6 = H3yx;c) S = H3": d ) 6 = H5". The signal labeled GP arises from E-selectin. This can be shown by comparison
with NOESY spectra of free E-selectin (not shown).
galactose protons differ
neuraininic acid residue. This supports the preferred gg
= gi/iic~hr,gazcc~/ze)
orientation at the C6N-C7" bond. With
regard to the orientation at the C7N-C8N bond, analysis of the
vicinal homo- and heteronuclear coupling constants and the
determination of the 3C spin-lattice relaxation times have
shown that in aqueous solution the hydroxyl groups attached to
the c7N-Cgh bondIl'". 151 are oriented exclusively trans and
that a conformational equilibrium is observed only for the hydroxymethyl group on C9. Hence, the glycerol side chain of the
N-;icetylnetirnminic acid is thought to assume an orientation
like that described in ref. [12a].
The transfer NOES observed between galactose and N-acetylglucosamine correspond to the NOEs in the free tetrasaccharide
(Fig. 1 ) . The interglycosidic transfer NOEs originating from
fiicose. on the other hand. differ considerably from the corresponding NOEs in the free tetrasaccharide. Only an extremely
weak transfer N O E is observed between H5F and H2" (Fig. 2d).
Similarly there is a slightly weaker transfer NOE between the
methyl group of the N-acetyl function on C2GN and H1"
(Fig. 221). A very weak transfer NOE between HI and H5C or
H9". which is marked in Figure 2a, most likely arises by spin
diffusion (based on consideration of the NOE buildup curves).
The intensity of the transfer NOE between CH,-6F-and H2". on
the other hand, resembles that of the corresponding NOE in the
free tetrasaccharide (Fig. 1). A transfer NOE is also detected
between HS'and Hh", which in sialyl Lewis' lies at the detection
limit (Fig. 2 d ) .
For further evaluation the transfer NOEs were integrated;"']
the intcnsities of the intraglycosidic transfer NOEs between
H3:, and H3L?qwere used as calibration values. The relative
N O E s were converted into distance restrictions (Table 2) by
Txhle 2 t.\perimci!tnll~ deteriiuned interglycosidr tranafer NOEs from the
NOESY \prcmin (1.1s. 1 h) used lor distance restrictions (The NOEs for the
NOESY \pecti-uni (Fig. 1 3 ) of the free tetrasaccharide are also given for comparison.] For the detel-~iiiii~ition
ot lhc distnnce restrictions. rd (lower limit) and r h (upper
liini~).c~ilibratio!i \\+is carried out with a known intraglycosldic distance.
H I ' H? = 1 3 A according to the "Isolated Spin Pair Approximation" (lSP.4)
[I(,]. For llic d ~ ~ ~ r i i i i i i i i tofthc
i o n NOEs il relatively large error of *40% w a s used.
The \ ~ a l i i c0 ~1 the di\t;iiice rcstrictioiib F., and r h were t h e n obtained from the dist i i i i w \ cnlciiI.itcd Iron1 thc NOES by means of ISPA and the estimated errors.
8 (-15)
- 3 1 ( - 41
-45 ( -20)
- .37 (0)
0 I - 4)
0 (0)
7 7
application of the ISPA approximation,[l'] and these in turn
were used to find the conformation of bound sialyl Lewis". For
this a 10"-step Metropolis Monte Carlo (MMC) simulation['81
with a temperature parameter of 2000 K was performed which
completely encompassed the conformational space available to
sialyl Lewis".['"' From the 457 309 conformations obtained, 29
structures were selected which fulfilled all the distance restrictions. The conformations thus found populate only a very restricted region of the conformational space and thus yield the
conformation of bound sialyl Lewis" (Fig. 3).
From their experiments Cooke et al. derived a conformation
in which the N-acetylneuraminic acid has an orientation similar
Fig. 3 . Representation of the conformation of sialyl Lewis" in the bound state. Ten
of the 29 conformations obtained are shown. The following average values were
obtained for the dihedral angles at the glycosidic bond (cf. Table?):
gN (, = -76 + I;- 10 , ~N (; = 6 i/- 1 0 . $ti(,h = 3 Y
-10 . $,, (;N =
12 +
6 . gl (ih = 38
7 . $1 (:N = 2 6
:- 6
1 -
to that described here. However, they neither observed nor discussed a transfer NOE between H s Nand H3G,which is an essential distance restriction for deriving the bound conformation.
The authors therefore used already published force field calculations" I h l and molecular dynamics (MD) simulations'' 31 to be
able to suggest a possible conformation for the bound ligand.
The use of the exclusion principle -only the missing NOE H3G/
H3:x is enlisted for an upper distance restriction- -favors the
conformation C suggested by Ichikawa et al." Ihl
Transfer NOEs
between the fucose and the N-acetylglucosamine unit were not
discussed, and as a result the fucose residue in sialyl Lewis" is
assigned the orientation present in aqueous solution. In the
recent publication of Hensley et al.['] a "transfer NOE" between
H3yx and H3" was found which, it is claimed. shows that the
conformation in the bound state is identical with the conformation of sialyl Lewis" in solution. It is not clear, however, which
solution conformation is meant. Probably conformations A or
B suggested by Ichikawa are implied.[' Ih1
This cannot be reconciled with the results of Cooke et al.'41 nor can it be correlated
with our results. Possibly the "transfer NOE" observed by these
authors[51may be explained by the use of a large excess ofligand
and the presence of unbound sialyl Lewis".
Our experiments enable the determination of the conformation of bound sialyl Lewis' on the basis of M M C simulations at
2000 K. The high temperature parameter was chosen in order to
encompass, as fully as possible, the sterically available conformational space. It should be emphasized that the orientations of
both the N-acetylneuraminic acid and the fucose residue in
bound ligands lead to transfer NOES that differ considerably
from those for free sialyl Lewis" (Figs. 1 and 2).
The most important conclusions are that E-selectin complexes exclusively a conformation of sialyl Lewis" from the conformational mixture in aqueous solution in which the neuraminic
acid shows an orientation corresponding approximately to conformation A of Rutherford et al.1131or conformation C of
Ichikawa et al.[llh]and in which fucose differs in it\ orientation
from that preferred in aqueous solution (cf. Fig. 3 ) . From more
precise evaluation of the transfer NOE data it is possible to
derive possible conclusions as to the flexibility of the ~ ( 1 3)glycosidic bond between the fucose and the N-acetylglucosamine residue. These results are used at present as a basis for
the modeling of potent inhibitors for E-selectin receptors.
Received. February 25. 10% [Z7735IE]
Gel-man version: Aiigcii . Chmr. 1995. / / / 7 ~2 0 3 4 ~?(I37
Keywords: carbohydrates glycoproteins . N M R spectroscopy .
selectins . sialyl Lewis'
[I] M . P. Bevilncqua. S. Stengelin. hl. A Gimhrone. J r . B. Seed. S I ~ I W I C1989.
343. 1160-1165
3, Presky. P.
an. J. M . Rumbcrsei-. S. Li. K:S.
Familletti. B. A. Wolitzk!. D. K Burns. .Voitrri,
lale. S . (i Lister. G. Shah. M P. Weir. B i o ~ / i w i i
33. 105'11 ~105')6.
151 P. Hcnslc>. P. J. McDevitt. I.Hrookc.J J Trill. J. 4. Feild. D. E. hlcNulty. J. R.
Connor. D. E Grisnold. N V. Kuniai-. K . D. Kopplc. S. A . C'arr. B. J. Dalton.
K , Johanson. .I &i(J/.
1994, 269. 21949 330%
[h] a1 ( i . M . Clorc. A M Gronenhorn. .I . M ~ i , ! y i .R c i i i i 1982. 48. 402 -417: h)
G. M. Chi-e. A. M GI-onenhorn.ihid 1983. 53. 423 ~442:
c ) F. Xi, .I , M q y
Rcwrio~i.1992. !Zi. 651 656
[7] €5. Ernst, N . Cooke. P M. ,&her?. unpublished. The qrithetic tetrasaccharide
corresponds t o thc formula shown in the text.
181 The cctndnm;iins of h u m a n E-selectiii were combined b! means of the polymera\echnin iretictimi (FCR) uith the Fc region of liuman I g G l , a n d subcloned
i i i t h e expression vector pcDNAl iieo (Invitrogen). Following trmsfection of
CHO-KI cells (ATCC CCL-61) and selection with '3418 (Gibco). :i stahle cell
line w s obtained w h i c h secreted c'r 25 pgni1. ' recombinant E-selectin IiIgG.
For thc productimi of larger aniouiit~t h i s cell line was cultivated in 3 hollo~r
fiber hioreactor (1.1 in2. 50 mL, Cellco) in Opli. MEM culture m e d i u m (Ciihco) rupplementcd with 2 % fetal calf \ei-um nnd 200 m L - ' gentamyciii. The
supernatant o l ~ o n d i t i o n ~c cdl h was lir5t purified by d h t y chromatography
on proteiii A agarme (Sigma). The proteiii fraction obtained hy elution with
20mM glqcine-HCl. pH 3.0. w a s iicutralired and chi-oin;itographed direct11
on an .inti-human-E-selectiii affinity column ( 3 mg monoclonal antibody 7A9
per mL affigel). Specifically horiiid E-selectin l g G was eluted nitli 5 v urea in
buffered s a l i solution and finally dialyzed against Dubeccos PBS solution
(PBS = phosphate buffered \aline). The protein thus obtained showed. i n SDS
(sodium dccyl sull'atc) polyacry1;rmidc pel electrophoresis tinder reducing coiiditions. :i single band :it ca 140 kD. For N M R triialyris c a 6 me E-selectin
hJgG wab diiilyxd against 50mM pcrdeutei-ated imidazol (Signs). I mh4 CaCI,. pH 7.4.iii D1O a n d concentrated by-means of Centricon YM-50 (Amicon)
t o il Sinal volume ofO.5 mL.
[O] a ) All NMR experiment.; w r e performed i n the Jnstitut fur Binpliqsikalisclie
. .
C;hemic (lei- Uniccrsitiit Fi-ankfiirt on :i Rruker DMXhOO specti-ometei-.We
~ i s hto tliaiik Prof. Dr. H. Ruterjans for the the opportunilq to make the
measurements. b) For the N MR eyperiments on the complex an E-selectin(1gG
c1iiiner:i)'retrasacchnride ratio of 1: 15 M R S chosen. The concentration ofsialyl
Letcis" \\>is 0.X1 i n h i . that of E-selectin(lgG chimera) 5 4 ~ [D,]Imidarolc
~ .
(30iiihr) m a s used as buffer. In addition the tolution contained N a C l (50mxi)
and CaCI, (1 nib<). The p H w a s 7.4. Spinlock filtered NOESY spectra [lo] with
mixing times T,,,of 25. 50. 75. 100. 125. 150. 175. and 200 ms were recorded at
600 MMr a n d 310 K . The relaxation lime way 1 X5 s. The spectral \bid111 was
i000 H r (5 ppiii) in all casea. 512 Increments i n T , Rere recorded. For each
iiicrcment 32 transients were xcqnired each with 2K data points. After Lero-filling and multiplicirti~~n
with squared coc functions i n r l and T?. ii 2K x 1 K data
matrix b a s obtaincd by 2D Fourier transformation.
T. Scherf. J Anglister. Biophr.~..I 1993. 64. 7.54- 761
NOESY specti-a o f lkcc F
ciin(JgC~chimera) and sialyl Lewis" tetracaccharide were recorded :is des
d i n ref 191 with the following clxinges. For free
E-selectin mixing times T," of5O. 100. l50.100. and 400 ms (\\it11spinlock filtei-)
were used: for free si:il)I L.eni.;' inisiiig times of 200. .TOO. 500. hOO. 800. and
900 nis (without spinloch filter) were used, For free sial?l Lahis' a mcasurement temperelure o S 303 K \>as chosen
a ) J. Brcg. L. 24. J. Krooii-Batenburg. G. Strccker. J. Montrcuil. J. F. G.
Vliegenthart. Eur. J Biochcn;. 1989. 178. 727- 739: b) Y.Ichikawa. Y:C. Lin.
D. P. Dumas. G:J. Shcn. E. Ciai-cia-Joiiceda, M. 4
Ketcham. I.. E. Wnlkcr. 1. C. Paulson. C.-H. Wong.
114. 9281 9298: c ) C. Muk1iopadhy;iy. K. E. Miller.
1994. 34. ? I -19
7 J. Rutherford. L). G. Spackmain. P. J. Simpson. S. W. Homans. G / w o / m / o , q
1994. 4. 59-68.
a) X . Miirali. G. K . .Jaron. S B. Land). B. D. N . Ruo. B i o d i e ~ n i r t r i ~
1993. 32. 12941 11948: 11) F Ni, H . A . Scheraga. 4 ( ~ .C/IWI. Rrc.
1994. 17. 257 ~ 2 h 4 .c) F. Ni. Y Zhu. J. 1 \ 4 q ~ .Rr.ron. S e r . B 1994. 102.
a) 1,. Poppe. R. Stuikc-Pi-ill. I3 Meyer. H van Malheek. J: Bioi~iol.) V M K 1992.
2, 109- 136; b) Ci. Zhu, .4. Renuick. A. Bax. J. . M c i p ~K c s ~ i i rS. c r A 1994, 1 1 0 .
257-261: c i 1. LV. Jaqnes. S. Glant. W. Weltner. Jr.. Cirr/wlii,dr.Res. 1980. 80.
207- 21 I: d) M. F. Crurniccki. F. R. Thornton. J 4177. Chr~117.
,Sm.1977. 99.
8273 827').
1iikegr;iiions of the NOF cross signal\ wcrc carried out uitli UXh;MR software
(Bruker. Kai-lsrulie). The spectra shnwii in Figure 1 uere selected for integration. The NOE cross signal betneeti H7>, iind H1:" w a s used :is II reference
( 1 0 0 " ~ n ) Trantfei- NOEs :ire roughly aii order of mac.nikudc more i n t e n w e
than NOES for fi-ee sial?l Le\\is'.
[I71 4 . 11 Gronenhoi-n. G . M Clore. Prop. .\:iiil. .Woar7. K c m i . .Speci;o,ii. 1985.
17. 1-32,
[I81 ;I) I Peters. B Meqer. R . Stuike-Prill. R. Somor.jai. .LR Briuson. Cirrhohi.r/r.
Rrs. 1993. 338. 49 73: b ) R. Stuikc-1'1-ill. B. Mryer. hi..I Bbdicm. 1990. /94.
'103- 919.
[ I 0 1 The M M C simulations for s i a l h l Lewis' nere carried oiit with ii tcmpcralure
parameter oS 2000K and 1 x 10" maci-ostcps. i h e maximum step sire \\a\ 20
(d. $1 a n d 25 (1,)). The dihedml angles were defined ;is follows: h = HI-C 101-CY (for NeiiNAc- CI-C2-02-Cx). I// = C 1 - 0 - C x - H x Ifor YeuNAc: C'?.
02-Cu-Hx). a n d 01 = 05-C'5-Ch-Oh (for NcuNAc corresponding decignationa
are wed to iiidicatc the orientation o f the side chain). Th
46%. The choice of I: high temperature parameter ensured that all rtericall?
possible conformations were conFidercd. The evaluation oflhc M M C data a n d
the extraction of the conformationr that satisfied the expel-irncntally dctermined distance irestrictions (see Table 2 ) were pcrformcd uitli Fortran and
MATL.4U programs. All calculations were performed o n ;I Silicon (;r;rphics
Jndy I I \\orkst:itioii
Palladacycles as Structurally Defined Catalysts
for the Heck Olefination of Chloro- and
Bromoarenes **
Wolfgang A. Herrmann." Christoph Brossmer,
Karl Ofele. Claus-Peter Reisinger, Thomas Priermeier.
Matthias Beller, and Hartmut Fischer
Drdicated l o Prqfcssor Hcnri Brunner
on rile occasiori of'kis 60th birthday
The Heck reaction has developed into a standard method of
organic synthesis since its discovery in 1971.['.'I With it, styrene
derivatives, amongst others, can be prepared as vinylic C--C
coupling products in one step from iodo- and bromoarenes.
Because the reaction is both regio- and stereoselective, it is often
used in heterocycle and natural product c h e i n i ~ t r y .The
' ~ ~ Heck
reaction is also beginning to make headway in polymer chemistry.["] However, attempts to submit the cheap chloroarenes to
the Heck olefination were not an unqualified success.[51We have
found new, structurally defined, easy-to-handle palladium complexes, that surpass all previously known catalysts of the Heck
reaction as regards stability and lifetime. They also enable the
activation of chloroarenes.
X = Br, I
[Pd] = Pd(CH,CO&/P(C,H&
B = Base, e.g. N(CzH5)3,KzC03, Na(CH3CQ2)
Prof. Dr. W. A. Herrmmn. Dr. C. Brosumer. Dr. K . 6l'clc. C -I' Reicinger.
T. Priermeier
Anoreanisch-cheniisches Institut der Technischen L'iiit ei \itat Mlinclieii
Lichtenbergstrassc 4. D-X5747 Garching IGeimian! )
Telefax: Itit. code + (89) 1209-3473
e-mail. herrmaiiiva za~~hod.anorgchemie.tu-niiicnclien.dc
Dr. M .Beller. Dr. H. FischciHoechst AG. Zentralforschunp. D-65926 Frankfurt a m M ; m (Germany)
I**]Coordination Chemistry and Mechanisms ol~Metal-Catal!red C C Coupling
Rcactions. Part 5. This woi-k was supported by the Fonds der Chemischen
lndustrie and the Deutsche Forschiingsgeinein\ch:i~t Part 1-Ref. [:a]
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conformational, complex, determination, carbohydrate, selectinsialyl, lewis, bioactive, ligand
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