PDBsum entry 1pwu

Hydrolase

PDB id: 1pwu

Contents

Protein chains

747 a.a.

Ligands

GM6 ×2

Metals

_ZN ×2

References listed in PDB file

Key reference

Title

The structural basis for substrate and inhibitor selectivity of the anthrax lethal factor.

Authors

B.E.Turk, T.Y.Wong, R.Schwarzenbacher, E.T.Jarrell, S.H.Leppla, R.J.Collier, R.C.Liddington, L.C.Cantley.

Ref.

Nat Struct Mol Biol, 2004, 11, 60-66. [DOI no: 10.1038/nsmb708]

PubMed id

14718924

Abstract

Recent events have created an urgent need for new therapeutic strategies to treat anthrax. We have applied a mixture-based peptide library approach to rapidly determine the optimal peptide substrate for the anthrax lethal factor (LF), a metalloproteinase with an important role in the pathogenesis of the disease. Using this approach we have identified peptide analogs that inhibit the enzyme in vitro and that protect cultured macrophages from LF-mediated cytolysis. The crystal structures of LF bound to an optimized peptide substrate and to peptide-based inhibitors provide a rationale for the observed selectivity and may be exploited in the design of future generations of LF inhibitors.

Figure 1.
Figure 1. Inhibition of LF by GM6001. (a) GM6001 inhibits cleavage of MKKs by LF in vitro. Immunoblots show LF cleavage of MKK-3 and MKK-1 in J774A.1 lysates in the presence of varying concentrations of GM6001 or 10 mM o-phenanthroline, a metal chelator. Cleavage of MKK-3 causes a mobility shift; the MKK-1 antibody is directed against the N terminus and does not react with the cleavage product, resulting in disappearance of the band upon cleavage. (b) GM6001 inhibits MKK-3 cleavage in lethal toxin -treated cells. Quantified western blot analysis of MKK-3 cleavage in J774A.1 treated with lethal toxin (0.5 g ml-1 PA with the indicated concentrations of LF) in the absence or presence of 100 M GM6001. (c) Protection of J774A.1 cells from lethal toxin -mediated cell death by GM6001. Cell viability as determined by MTT assay after lethal toxin treatment in the presence of 100 M GM6001 or 0.2% (v/v) DMSO carrier. (d) Dose-dependent neutralization of lethal toxin by GM6001. J774A.1 cell viability determined by MTT assay after treatment with lethal toxin (0.5 g ml-1 PA + 0.3 g ml-1 LF) or PA alone (0.5 g ml-1) in the presence of the indicated concentrations of GM6001. (e) GM6001 protects J774A.1 cells when added subsequent to LeTx. Cell viability is shown after treatment with PA alone (0.4 g ml-1) or PA with LF (25 ng ml-1), with GM6001 added to 100 M at the indicated time after toxin addition.

Figure 2.
Figure 2. Structures of LF in complex with peptides and inhibitors. Molecular surface of LF is colored by charge (red, negative; blue, positive), with Zn2+ as a solid sphere (cyan) and the model of the peptide or inhibitor in ball-and-stick representation. The individual electron density surrounding each molecule is a 2F[o] - F[c] difference map calculated at the respective final resolution and contoured at 1.0 . (a) LF20 (yellow) in the absence of Zn2+, resolution limit 2.85 Å. The model of bound LF20 shows the sequence VYPYPMEPT (residues 8 -16 of the 20-residue-long LF20). This is the ordered region, and the electron density is clearly visible in difference maps (2F[o] - F[c] and F[o] - F[c]) calculated from crystal X-ray diffraction data. (b,c) SHAc-YPM (white, labeled YPM), resolution limit 3.50 Å, and GM6001 (green), resolution limit 2.70 Å, respectively. Continuous electron density extends from the zinc atom to the metal-chelating moieties of the inhibitors (hydroxamate and thioacetyl, respectively). (d) The superposed individual complex structures of all three target molecules from a -c in the substrate-binding groove of LF, using the surface calculated for LF -LF20. The targets are all bound in the same N-to-C peptide orientation. (e) An overview of LF bound to the targets LF20, GM6001 and SHAc-YPM, superposed and colored as in d. The molecular surface was calculated from the LF -LF20 complex. The domains in LF are labeled I -IV. The catalytic site is in domain IV, where the zinc atom (not shown in this figure) is bound. These figures were prepared using SPOCK (http://mackerel.tamu.edu/spock/).

The above figures are reprinted by permission from Macmillan Publishers Ltd: Nat Struct Mol Biol (2004, 11, 60-66) copyright 2004.