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PDBsum entry 1fd7
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Contents |
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* Residue conservation analysis
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References listed in PDB file
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Key reference
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Title
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Exploration of the gm1 receptor-Binding site of heat-Labile enterotoxin and cholera toxin by phenyl-Ring-Containing galactose derivatives.
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Authors
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E.Fan,
E.A.Merritt,
Z.Zhang,
J.C.Pickens,
C.Roach,
M.Ahn,
W.G.Hol.
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Ref.
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Acta Crystallogr D Biol Crystallogr, 2001,
57,
201-212.
[DOI no: ]
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PubMed id
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Abstract
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Cholera toxin (CT) and the closely related heat-labile enterotoxin of
Escherichia coli (LT) are responsible for numerous cases of diarrhea worldwide,
leading to considerable morbidity and mortality. The B subunits of these
heterohexameric AB(5) toxins form a pentameric arrangement which is responsible
for binding to the receptor GM1 of the target epithelial cells of the host.
Blocking these B pentamer-receptor interactions forms an avenue for therapeutic
intervention. Here, the structural characterization of potential
receptor-blocking compounds are described based on the previously identified
inhibitor m-nitrophenyl-alpha-D-galactoside (MNPG). The structure of a CTB-MNPG
complex confirms that the binding mode of this inhibitor is identical in the two
homologous toxins CT and LT and is characterized by a glycosyl linkage geometry
that leads to displacement of a well ordered water molecule near the amide group
of Gly33 by the O1-substituent of MNPG. This glycosyl geometry is not maintained
in the absence of a substituent that can displace this water, as shown by a
complex of LTB with p-aminophenyl-alpha-D-galactoside (PAPG). New compounds were
synthesized to investigate the feasibility of maintaining the favorable binding
interactions exhibited by MNPG while gaining increased affinity through the
addition of hydrophobic substituents complementary to either of two hydrophobic
regions of the receptor-binding site. The structural characterization of
complexes of LTB with two of these compounds,
3-benzylaminocarbonylphenyl-alpha-D-galactoside (BAPG) and
2-phenethyl-7-(2,3-dihydrophthalazine-1,4-dione)-alpha-D-galactoside (PEPG),
demonstrates a partial success in this goal. Both compounds exhibit a mixture of
binding modes, some of which are presumably influenced by the local packing
environment at multiple crystallographically independent binding sites. The
terminal phenyl ring of BAPG associates either with the phenyl group of Tyr12 or
with the hydrophobic patch formed by Lys34 and Ile58. The latter interaction is
also made by the terminal phenyl substituent of PEPG, despite a larger ring
system linking the galactose moiety to the terminal phenyl. However, neither
BAPG nor PEPG displaces the intended target water molecule. Both of the designed
compounds exhibit increased affinity relative to the galactose and to PAPG
notwithstanding the failure to displace a bound water, confirming that
additional favorable hydrophobic interactions can be gained by extending the
starting inhibitor by a hydrophobic tail. The insight gained from these
structures should allow the design of additional candidate inhibitors that
retain both the glycosyl geometry and water displacement exhibited by MNPG and
the favorable hydrophobic interactions exhibited by BAPG and PEPG.
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Figure 2.
Figure 2 MNPG binding by cholera toxin. Superposition of the
binding site of MNPG complexed with the B pentamer of two
homologous toxins, LT and CT. The LTB-MNPG complex (green, PDB
code [109]1lt6 ; Merritt et al., 1997[110] [Merritt, E. A.,
Sarfaty, S., Feil, I. K. & Hol, W. G. J. (1997). Structure, 5,
1485-1499.]-[111][bluearr.gif] ) was superimposed onto the new
structure of the CTB-MNPG complex (blue) by least-squares
minimization of the coordinate difference for atoms C, N, CA, O,
CB of conserved residues within 10 Å of one of the five
independent binding sites (site F in this figure). The r.m.s.
coordinate difference for the 157 superimposed atoms at this
site was 0.30 Å. The only sequence difference between the two
toxins in the immediate vicinity of the binding site is at
residue 13, which is not involved in binding MNPG. (a)
Electron-density contours at 4 [112][sigma] are shown from a
[113][sigma] [A]-weighted omit map (mF[o] - F[c]) of the
CTB-MNPG complex at 2.0 Å resolution; the five copies of the
ligand and all waters within 8 Å were omitted from the
calculation of F[c]. (b) Water molecules identified near all
five binding sites have been superimposed back onto site F.
Consensus water sites #1, #3 and #5 of the receptor-binding site
are labeled (numbering as in Merritt, Sixma et al., 1994[114]
[Merritt, E. A., Sixma, T. K., Kalk, K. H., van Zanten, B. A. M.
& Hol, W. G. J. (1994). Mol. Microbiol. 13,
745-753.]-[115][bluearr.gif] ). The position of water Wat2 is
occupied by O2' of the nitrophenyl group.
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Figure 3.
Figure 3 PAPG binding to heat-labile enterotoxin. Stereo pair
showing the binding mode of PAPG to the LT B pentamer. The
protein (green), ball and stick PAPG molecule and 2.5  electron-density
contours are shown for a single binding site (chain D) of the
LTB-PAPG complex. Water molecules (cyan) and the PAPG molecule
(red) from the four other copies of the binding site in the
present structure are shown superimposed onto this single site
to indicate the degree of reproducibility of water placement and
the range of conformations seen for the aminophenyl moiety of
PAPG. The binding mode of MNPG as seen in the LTB-MNPG complex
(Merritt et al., 1997[Merritt, E. A., Sarfaty, S., Feil, I. K. &
Hol, W. G. J. (1997). Structure, 5, 1485-1499.]) is shown
superimposed in gold. Electron density is from an (mF[o] - F[c])
map at 1.6 Å resolution in which all ligand molecules and all
water molecules within 8 Å of the binding site were omitted
from the calculation of F[c].
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The above figures are
reprinted
by permission from the IUCr:
Acta Crystallogr D Biol Crystallogr
(2001,
57,
201-212)
copyright 2001.
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