PDBsum entry 1fd7

Toxin

PDB id: 1fd7

Contents

Protein chains

(+ 4 more) 103 a.a.

Ligands

AI1 ×10

Waters ×856

PDB id:

1fd7

Name:

Toxin

Title:

Heat-labile enterotoxin b-pentamer with bound ligand bmsc001

Structure:

Heat-labile enterotoxin b chain. Chain: d, e, f, g, h, l, m, n, o, p. Engineered: yes

Source:

Escherichia coli. Organism_taxid: 562. Expressed in: escherichia coli. Expression_system_taxid: 562.

Biol. unit:

Pentamer (from PQS)

Resolution:

1.80Å R-factor: 0.187 R-free: 0.234

Authors:

E.Fan,E.A.Merritt,J.Pickens,M.Ahn,W.G.J.Hol

Key ref:

E.Fan et al. (2001). Exploration of the GM1 receptor-binding site of heat-labile enterotoxin and cholera toxin by phenyl-ring-containing galactose derivatives. Acta Crystallogr D Biol Crystallogr, 57, 201-212. PubMed id: 11173465 DOI: 10.1107/S0907444900016814

Date:

19-Jul-00 Release date: 10-Aug-00

Protein chains

P32890  (ELBP_ECOLX) -  Heat-labile enterotoxin B chain from Escherichia coli

Seq: 124 a.a.

Struc: 103 a.a.

DOI no: 10.1107/S0907444900016814

Acta Crystallogr D Biol Crystallogr 57:201-212 (2001)

PubMed id: 11173465

Exploration of the GM1 receptor-binding site of heat-labile enterotoxin and cholera toxin by phenyl-ring-containing galactose derivatives.

E.Fan, E.A.Merritt, Z.Zhang, J.C.Pickens, C.Roach, M.Ahn, W.G.Hol.

ABSTRACT

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.

Selected figure(s)

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.

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].

The above figures are reprinted by permission from the IUCr: Acta Crystallogr D Biol Crystallogr (2001, 57, 201-212) copyright 2001.

Figures were selected by an automated process.