by Thiang Yian Wong, Robert Schwarzenbacher and Robert C. Liddington
The Burnham Institute, 10901 N Torrey Pines Rd, La Jolla, CA 92037,
Anthrax Toxin, together with its bacterial capsule, is a major virulence factor
in Anthrax1. The virulent strain of Bacillus
anthracis is an encapsulated gram-positive, rod-shaped, spore-forming
bacterium that produces and exports the three Anthrax Toxin proteins,
Protective Antigen (PA), Lethal Factor (LF) and Edema Factor (EF). The
infectious agent of anthrax, the bacillus spore, may be introduced into a
mammalian host through inhalation, ingestion and subcutaneous wounds. Once in
a susceptible host organism, the spores germinate within macrophages and their
vegetative cells proliferate rapidly2. The
explosive release of bacilli from the host macrophages
simultaneously introduces the three toxin components into the bloodstream.
Continuous rapid bacterial proliferation and toxin production by the bacilli
could lead to very high concentrations of toxin accumulation in the
bloodstream. Although antibiotics may be effective in clearing the bacteria
from the bloodstream, high level of toxins may remain in the blood circulation
for a further three to four days. In the bloodstream, PA attaches to the host
cell surface via the anthrax toxin receptor (ATR)3.
Furin, a cell surface protease, then removes a 20kD fragment of PA and
initiates the formation of the pore-forming complex of the anthrax toxin. Seven
copies of the processed PA (now a 63 kD form of PA, PA63), together
with seven copies of the ATR, form a seven-membered ring4. The PA63
heptamers combine with either of two enzymes, LF (a zinc metalloprotease) or EF
(an adenylate cyclase), to form either of the two anthrax toxins, Lethal Toxin
(LeTx) or Edema Toxin (EdTx). The whole toxin assembly is taken into an
intracellular compartment through "endocytosis". Acidification within the
endosome through natural cell processes triggers a conformational change in the
PA63 heptamer, causing it to form a channel that punctures through the membrane
of the endosome and facilitates the translocation of the active enzymes into
the host cell cytosol (interior). LeTx is specifically cytotoxic towards the
host's white blood cells called macrophages, with LF targeting five of seven
members of the Mitogen-Activated Protein Kinase Kinase (MAPKK) family5, a
protein family involved in the cell signaling essential for normal cell growth
and differentiation. Proteolysis of the MAPKKs at their N-termini by LF rapidly
blocks signals to recruit other immune cells to fight the bacterial
EF, which is a calmodulin-activated adenylyl cyclase, can increase the
concentration of adenosine 3',5'-cyclic monophosphate (cyclic AMP or cAMP), a
secondary messenger that regulates cell function inside the host cell, to
abnormal levels7. Accumulation of fluids within and
between cells occurs due to this abnormality, leading to edema. In advanced
anthrax intoxication, especially in highly fatal cases of inhalation anthrax
where LF is the greatest source of damage, an antitoxin such as an inhibitor
against LF, would be critical as part of a combined therapy with other existing
treatments (eg. antibiotics, anti-PA antibodies, critical care).
Figure 1. Crystal structures of LF-LF20, LF-thioacetylYPM-zinc, and
The molecular surface of LF is colored by charge (red = negative; blue =
positive), with Zn2+ shown as a solid sphere (cyan) and the model of
the peptide or inhibitor shown in stick representation. (A) The superposed
individual complex structures of the three target molecules: LF20 (yellow gold)
in the absence of Zn2+, resolution limit 2.85 Å; SHAc-YPM (tinted
white, labeled "YPM"), resolution limit 3.50 Å; and GM6001 (green), resolution
limit 2.70 Å. The model of bound LF20 shows the sequence VYPYPMEPT (residues 8
to 16 of the 20-residue long LF20). This was the ordered region, and the
electron density was clearly visible in difference maps
(2Fo-Fc and Fo-Fc) calculated from
crystal X-ray diffraction data. All three molecules are shown bound 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. (B) An overview
of LF bound to the targets LF20, GM6001, and SHAc-YPM, superposed and colored
as in (A). The molecular surface was calculated from the LF-LF20 complex. The
domains in LF are labeled I, II, III and IV. The catalytic site is in Domain
IV, where the zinc atom (not seen in this figure) is bound. These figures were
prepared using SPOCK14.
To date, the crystal structures of all three proteins, PA4,
LF8 and EF9 that
make up the Anthrax Toxin components have been solved. Previously, we had
reported the crystal structure of LF, and LF bound to the N-terminus fragment
of its natural substrate, MAPKK28 (also see SSRL
Science Highlights, April
2002). In collaboration with researchers at Harvard Medical School and United
States Army Medical Research Institute of Infectious Diseases (USAMRIID), we
have now obtained crystal structures of LF bound to an optimized substrate
peptide and also of LF bound to several small molecule inhibitors10, 11. Data
collection from the protein crystals was done at the Stanford Synchrotron
Radiation Laboratory (SSRL), Menlo Park, California, on the macromolecular
crystallography Beamlines 9-1 and 7-1, and also at the National Synchrotron
Light Source (NSLS), Brookhaven, New York.
Figure 2. X-ray crystal structure of the LF-NSC 12155-Zn complex.
(A) Molecular surface of LF colored by charge (red = negative; blue =
positive), with Zn2+ as a solid sphere in cyan. The model of the
inhibitor molecule NSC 12155 (carbons are in light blue), and an optimized
peptide with the sequence VYPYPME in light yellow, both shown in stick
representation. The inhibitor NSC 12155 binds to LF in the active site at the
hydrophobic neck of the pocket, where Tyrosine 4 of sequence VYPYPME
(equivalent of Tyrosine 11 of LF20) is highly selected as the optimized residue
to bind in this position. The binding position of NSC 12155 overlaps that of
the optimized peptide sequence at residues 2 and 3 (YP). This figure was
prepared using SPOCK14.
(B) The inhibitor NSC 12155 bound in the active site of LF. The difference map,
2Fo-Fc, calculated at 2.9-Å resolution limit, is
contoured at 1.0 s. A portion of NSC 12155 appears
non-rigid owing to a rotatable bond, with patchy electron density trace at 1.0
s level, although almost full electron density
coverage is seen for this portion at a contour level of 0.6 s. Inhibitor molecule (light blue), zinc-coordinating
residues (H686, H690, E735) and catalytic residues (E687, Y728) are in stick
representation. The Ca atoms of residues 680-694
(green, background) and 726-742 (beige, foreground) are in ribbon
representation. The Zn2+ ion (cyan) is a lined sphere, and its
with His686, His690 and Glu735 are represented as aligned small white spheres.
These figures were prepared using SPOCK14
In the work with colleagues at Harvard Medical School, the structural basis for
LF target recognition was investigated initially through finding an optimized
substrate for LF10. Two sets of peptide libraries
with randomized amino acid sequence in 13 residue-long peptides were generated,
with either the sequence N-terminal or C-terminal to the peptide cleavage site
randomized. Each residue that contributed to increased substrate catalysis rate
(increased turnover) at the individual specific position on the peptide
substrate was scored. A consensus peptide sequence was built upon these results
and we designed an optimized peptide substrate for further experimental work. A
20-residue long peptide was synthesized, using the consensus 13-residue
sequence identified, flanked by the amino acid sequence found in the natural
substrate for LF, MAPKK2. This peptide was named LF20 (MLARRKKVYPYPMEPTIAEG).
LF20 provided a probe to illustrate the shape, extent and limits of the
substrate-binding and catalytic site of LF. Based on the consensus sequence, a
small peptidic inhibitor named SHAc-YPM, consisting of a zinc-binding
thioacetyl moiety coupled to the three residues C-terminal to the cleavage site
in LF20, was also created (Ki = 11 ± 3 µM). Looking at
other known metalloprotease inhibitors with similar peptidic sequences to
SHAc-YPM, GM600112, 13, a
commercially available potent inhibitor of matrix metalloproteases (MMPs, a
family of cell surface proteins in mammalian cells implicated in cancer tumour
growth), MMP1 and MMP2, was assayed for and showed significant inhibition
towards LF (Ki = 2.1 ± 0.2 µM).
Simultaneously, in collaboration with colleagues at USAMRIID, who performed a
search for an effective LF inhibitor in a high-throughput format, using the
National Cancer Institute (NCI) Diversity Set
several candidate small molecule compounds were identified11 and set up for
LF-inhibitor complex crystals. One LF-inhibitor complex that gave the best
crystal data was that of LF bound to the hit, NSC 12155, a quinolone compound
that gave the highest inhibition constant in this screen
(Ki = 0.5
± 0.18 µM).
Crystal structures of LF-LF20, in the absence and presence of zinc10, and LF-SHAc-YPM10,
LF-GM600110 and LF-NSC1215511, all in the presence of zinc, were obtained to explore the
three-dimensional relationship between LF and its binding targets. The striking
features that were evident from the crystal structures were that all of the
peptides and small molecule inhibitors recognized and bound to LF via the
hydrophobic substrate-binding groove in the immediate vicinity of the catalytic
zinc. LF20 induced a conformational change in the active site of LF, and
Tyr11, when bound in the S1' hydrophobic pocket in
LF causes the pocket to expand, in a phenomena termed "induced fit". This would
not have been known if we did not have the consensus peptide LF20 to probe the
binding site of LF, and thus presents a novel look at the elasticity of LF in
its active site. The small molecule inhibitors on the other hand, provided us
with an indication of the criteria for inhibitor design, which guides us to
conclude that the main determinants for most effective inhibition is through
zinc-deprivation by a strong metal-chelating moiety, like the hydroxamate
group, coupled to a hydrophobic and stereochemically well-fitted small molecule
structure. The structure of LF-NSC12155 with11 and
without zinc (unpublished data), indicates that it may even be sufficient to
have a compound with hydrophobic interactions alone (without metal chelation)
to effectively inhibit the catalytic activity of LF.
Due to the diffraction limits and large unit cell dimensions of the protein
crystals possibly grown for LF, data collection at a synchrotron facility like
SSRL greatly enhances our ability to obtain data of high quality and resolution
to achieve the results for our studies. Crystal structures will be highly
important to guide future drug-designing efforts for LF inhibitors. The
continuing work on the inhibitors for LF will be towards generating chemical
derivatives with better inhibitory capabilities and cell permeability for
effective delivery of the potential drug molecule to the centre of LF activity
in the event of anthrax intoxication. We are at present working on this aspect
of our research, and will be frequent users of the SSRL for its synchrotron
Leppla SH in Comprehensive Sourcebook of Bacterial Protein Toxins 2nd
edn (eds Alouf JA & Freer J) 243-263 (Academic, London, 1999).
Hanna PC, Acosta D & Collier RJ. On the role of macrophages in anthrax.
Proc. Natl. Acad. Sci. USA 90, 10198-10201 (1993).
Bradley KA, Mogridge J, Mourez M, Collier RJ & Young JAT.
Identification of the cellular receptor for anthrax toxin. Nature
Petosa C, Collier RJ, Klimpel KR, Leppla SH & Liddington RC. Crystal
structure of the anthrax toxin protective antigen. Nature 385, 833-838 (1997).
Vitale G, Bernadi L, Napolitani G, Mock M & Montecucco C.
Susceptibility of mitogen-activated protein kinase kinase family members to
proteolysis by anthrax lethal factor. Biochem. J. 352, 739-745
Duesbury NS, Webb CP, Leppla SH, Gordon VM, Klimpel KR, Copeland TD,
Ahn NG, Oskarsson AK, Fukasawa K, Paull KD & Vande Woude GF. Proteolytic
inactivation of MAP-kinase-kinase by anthrax lethal factor. Science
280, 734-737 (1998).
- Leppla SH. Anthrax toxin edema factor: a bacterial
adenylate cyclase that increases cyclic AMP concentrations of eukaryotic cells.
Proc. Natl. Acad. Sci. USA 79, 3162-3166 (1982).
Pannifer AD, Wong TY, Schwarzenbacher R, Renatus M, Petosa C,
Bienkowska J, Lacy DB, Collier RJ, Park S, Leppla SH, Hanna P & Liddington RC.
Crystal structure of the anthrax lethal factor. Nature 414,
Drum CL, Yan S-Z, Bard J, Shen Y-Q, Lu D, Soelaiman S, Grabarek Z, Bohm A &
Tang W-J. Structural basis for the activation of anthrax adenylyl cyclase
exotoxin by calmodulin. Nature 415, 396-402 (2002).
Turk BE and Wong TY, Schwarzenbacher R, Jarrell ET, Leppla SH, Collier RJ,
Liddington RC & Cantley LC. The structural basis for substrate and inhibitor
selectivity of the anthrax lethal factor. Nat. Struct. Mol. Biol.
11, 60-66 (2004).
Panchal RG, Hermone AR, Nguyen TL, Wong TY, Schwarzenbacher R,
Schmidt J, Lane D, McGrath C, Turk BE, Burnett J, Aman MJ, Little S, Sausville
EA, Zaharevitz DW, Cantley LC, Liddington RC, Gussio R & Bavari S.
Identification of small molecule inhibitors of anthrax lethal factor.
Nat. Struct. Mol. Biol. 11, 67-72 (2004).
Grobelny D, Poncz L & Galardy RE. Inhibition of human skin fibroblast
collagenase, thermolysin, and Pseudomonas aeruginosa elastase by
peptide hydroxamic acids. Biochemistry 31, 7152-7154 (1992).
Levy DE, Lapierre F, Liang W, Ye W, Lange CW, Li X, Grobelny D, Casabonne M,
Tyrrell D, Holme K, Nadzan A & Galardy RE. Matrix metalloproteinase
inhibitors: a structure-activity study. J. Med. Chem. 41,
Christopher JA. SPOCK: The Structural Properties Observation and
Calculation Kit [program manual] (The Center for Macromolecular Design, Texas
A&M University, College Station, TX, 1998).