U.S. patent application number 09/821819 was filed with the patent office on 2002-07-18 for n-tradd active site and uses thereof.
Invention is credited to Hsu, Sang, Lin, Lih-Ling, Malakian, A. Karl, McDonagh, Thomas, Telliez, Jean-Baptiste, Tsao, Desiree H.H., Xu, Guang-Yi.
Application Number | 20020094540 09/821819 |
Document ID | / |
Family ID | 26890929 |
Filed Date | 2002-07-18 |
United States Patent
Application |
20020094540 |
Kind Code |
A1 |
Tsao, Desiree H.H. ; et
al. |
July 18, 2002 |
N-TRADD active site and uses thereof
Abstract
The present invention relates to the three dimensional solution
structure of the N-terminal domain of TNFR-1 associated death
domain protein ("N-TRADD"), as well as the identification and
characterization of a C-TRAF2 binding active site of N-TRADD. Also
provided for by the present invention are methods of utilizing the
three dimensional structures for the design and selection of potent
and selective inhibitors of TNF signaling pathways.
Inventors: |
Tsao, Desiree H.H.;
(Belmont, MA) ; Telliez, Jean-Baptiste; (Waltham,
MA) ; McDonagh, Thomas; (Acton, MA) ; Lin,
Lih-Ling; (Concord, MA) ; Hsu, Sang;
(Lexington, MA) ; Xu, Guang-Yi; (Medford, MA)
; Malakian, A. Karl; (Boxborough, MA) |
Correspondence
Address: |
Craig J. Arnold, Esq.
Amster, Rothstein & Ebenstein
90 Park Avenue
New York
NY
10016
US
|
Family ID: |
26890929 |
Appl. No.: |
09/821819 |
Filed: |
March 29, 2001 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60195370 |
Apr 6, 2000 |
|
|
|
Current U.S.
Class: |
435/7.1 ;
435/226; 702/19 |
Current CPC
Class: |
C07K 14/7151 20130101;
C07K 2299/00 20130101; G01N 33/6863 20130101; G01N 33/6803
20130101; G16B 15/30 20190201; G16B 15/00 20190201; G01N 2500/04
20130101 |
Class at
Publication: |
435/7.1 ; 702/19;
435/226 |
International
Class: |
G01N 033/53; G06F
019/00; G01N 033/48; G01N 033/50; C12N 009/64 |
Claims
What is claimed is:
1. A solution comprising an N-terminal domain of TNFR-1 associated
death domain protein (N-TRADD).
2. The solution of claim 1, wherein the N-terminal domain of TNFR-1
associated death domain protein comprises amino acid residues 1-169
of FIG. 1.
3. The solution of claim 2, comprising between 0.8-1.0 mM N-TRADD
in a buffer comprising 20 mM imidazole, 200 mM NaCl, 20 mM DTT and
0.05% NaN.sub.3, in either 90% H.sub.2O/10% D.sub.2O or 100%
D.sub.2O.
4. The solution of claim 3, wherein the N-TRADD is either
unlabeled, .sup.15N enriched or .sup.15N,.sup.13C enriched.
5. The solution of claim 4, wherein the N-TRADD is biologically
active.
6. The solution of claim 1, wherein the secondary structure of
N-TRADD comprises four beta strands forming an antiparallel beta
sheet, with five alpha helices packing around the beta sheet.
7. The solution of claim 6, wherein the beta strands and alpha
helices are configured in the trace order .beta.1, .alpha.1,
.alpha.2, .beta.2, .beta.3, .alpha.3, .beta.4, .alpha.4 and
.alpha.5.
8. The solution of claim 7, wherein .beta.1 comprises amino acid
residues S14-E20 of N-TRADD, .alpha.1 comprises amino acid residues
L28-Y32 of N-TRADD, .alpha.2 comprises amino acid residues P35-G53
of N-TRADD, .beta.2 comprises amino acid residues Q60-R66 of
N-TRADD, .beta.3 comprises amino acid residues L71-R76 of N-TRADD,
.alpha.3 comprises amino acid residues R80-L107 of N-TRADD, .beta.4
comprises amino acid residues Q115-R119 of N-TRADD, .alpha.4
comprises amino acid residues E132-A141 of N-TRADD and .alpha.5
comprises amino acid residues E150-N161 of N-TRADD.
9. An active site of a C-TRAF2 binding protein or peptide, wherein
said active site is characterized by a three dimensional structure
comprising the relative structural coordinates of amino acid
residues Y16, F18, and H65 according to FIG. 2, .+-.a root mean
square deviation from the conserved backbone atoms of said amino
acids of not more than 1.5 .ANG..
10. The active site of claim 9, wherein the three dimensional
structure of said active site further comprises the relative
structural coordinates of amino acid residues L17, V58, L59, I72,
and D149 according to FIG. 2, .+-.a root mean square deviation from
the conserved backbone atoms of said amino acids of not more than
1.5 .ANG..
11. The active site of claim 10, wherein the three dimensional
structure of said active site further comprises the relative
structural coordinates of amino acid residues K63, I64, D68, Q70,
V73, Q74, L75, C78, L118, G121, A122, R124, L125, E150, and L152
according to FIG. 2, .+-.a root mean square deviation from the
conserved backbone atoms of said amino acids of not more than 1.5
.ANG..
12. The active site of claim 10, wherein the root mean square
deviation from the conserved backbone atoms of said amino acids is
not more than 1.0 .ANG..
13. The active site of claim 10, wherein the root mean square
deviation from the conserved backbone atoms of said amino acids is
not more than 0.5 .ANG..
14. An agent which binds to the active site of claim 9, wherein
said agent is an inhibitor of TRADD function.
15. The agent of claim 14, wherein said agent is a protein,
peptide, nucleic acid or compound.
16. An agent which binds to the active site of claim 10, wherein
said agent is an inhibitor of TRADD function.
17. The agent of claim 16, wherein said agent is a protein,
peptide, nucleic acid or compound.
18. An agent which binds to the active site of claim 11, wherein
said agent is an inhibitor of TRADD function.
19. The agent of claim 18, wherein said agent is a protein,
peptide, nucleic acid or compound.
20. A method for identifying an agent that interacts with N-TRADD,
comprising the steps of: (a) determining an active site of N-TRADD
from a three dimensional structure of N-TRADD; and (b) performing
computer fitting analyses to identify an agent which interacts with
said active site.
21. The method of claim 20, wherein the active site comprises the
relative structural coordinates of amino acid residues Y16, F18,
and H65 according to FIG. 2, .+-.a root mean square deviation from
the conserved backbone atoms of said amino acids of not more than
1.5 .ANG..
22. The method of claim 21, wherein the active site further
comprises the relative structural coordinates of amino acid
residues L17, V58, L59, I72, and D149 according to FIG. 2, .+-.a
root mean square deviation from the conserved backbone atoms of
said amino acids of not more than 1.5 .ANG..
23. The method of claim 22, wherein the active site further
comprises the relative structural coordinates of amino acid
residues K63, I64, D68, Q70, V73, Q74, L75, C78, L118, G121, A122,
R124, L125, E150, and L152 according to FIG. 2, .+-.a root mean
square deviation from the conserved backbone atoms of said amino
acids of not more than 1.5 .ANG..
24. The method of claim 21, further comprising contacting the
identified agent with a molecule or molecular complex comprising
N-TRADD in order to determine the effect the agent has on said
molecule or molecular complex.
25. The method of claim 24, wherein the agent is an inhibitor of
the molecule or molecular complex comprising N-TRADD.
26. An agent identified by the method of claim 25.
27. A method for identifying an agent which is a potential
inhibitor of N-TRADD binding to C-TRAF2, comprising the steps of:
(a) determining an N-TRADD binding active site of C-TRAF2 from a
three dimensional structure of N-TRADD and a three dimensional
structure of C-TRAF2; (b) selecting or designing a candidate
inhibitor of N-TRADD binding to C-TRAF2 by performing computer
fitting analyses with the three dimensional structures of (a); and
(c) obtaining or synthesizing the candidate inhibitor.
28. The method of claim 27, comprising the additional step of
contacting the candidate inhibitor with N-TRADD and C-TRAF2 in
solution in order to determine the effect of the candidate
inhibitor on N-TRADD binding to C-TRAF2.
29. The method of claim 27, wherein the N-TRADD binding active site
of C-TRAF2 comprises the relative structural coordinates of amino
acid residues R393, Y395, D399, G400, F410, F447, R448, P449, D450,
S453, S454, S455, I465, A466, S467, G468, and P470 according to the
atomic coordinates specified in Accession Nos. 1CA4, 1CA9 or 1QSC
of the Protein Data Bank, .+-.a root mean square deviation from the
conserved backbone atoms of said amino acids of not more than 1.5
.ANG..
30. A method for identifying a potential inhibitor of N-TRADD,
comprising the steps of: (a) using a three dimensional structure of
N-TRADD as defined by the relative structural coordinates of the
amino acids of FIG. 2, .+-.a root mean square deviation from the
conserved backbone atoms of said amino acids of not more than 1.5
.ANG.; (b) employing said three-dimensional structure to design or
select a potential inhibitor; and (c) synthesizing or obtaining
said potential inhibitor.
31. The method according to claim 30, wherein the potential
inhibitor is designed de novo.
32. The method according to claim 30, wherein the potential
inhibitor is designed from a known inhibitor.
33. The method of claim 31, further comprising the step of
contacting the potential inhibitor with N-TRADD in the presence of
a binding protein to determine the ability of the potential
inhibitor to inhibit N-TRADD.
34. The method of claim 32, further comprising the step of
contacting the potential inhibitor with N-TRADD in the presence of
a binding protein to determine the ability of the potential
inhibitor to inhibit N-TRADD.
35. The method according to claim 30, wherein the step of employing
the three dimensional structure to design or select the potential
inhibitor comprises the steps of: (a) identifying chemical entities
or fragments capable of associating with N-TRADD; and (b)
assembling the identified chemical entities or fragments into a
single molecule to provide the structure of the potential
inhibitor.
36. The method according to claim 35, wherein the potential
inhibitor is designed de novo.
37. The method according to claim 35, wherein the potential
inhibitor is designed from a known inhibitor.
38. The method of claim 36, further comprising the step of
contacting the potential inhibitor with N-TRADD in the presence of
a binding protein to determine the ability of the potential
inhibitor to inhibit N-TRADD.
39. The method of claim 37, further comprising the step of
contacting the potential inhibitor with N-TRADD in the presence of
a binding protein to determine the ability of the potential
inhibitor to inhibit N-TRADD.
40. An inhibitor identified or designed by the method of claim
31.
41. An inhibitor identified or designed by the method of claim 35.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/195,370 filed Apr. 6, 2000.
FIELD OF THE INVENTION
[0002] The present invention relates to the three dimensional
solution structure of the N-terminal domain of TNFR-1 associated
death domain protein ("N-TRADD"), as well as the identification and
characterization of a C-TRAF2 binding active site of N-TRADD. These
structures are critical for the design and selection of potent and
selective inhibitors of TNF signaling pathways.
BACKGROUND OF THE INVENTION
[0003] Tumor Necrosis Factor (TNF) is a pro-inflammatory cytokine
that is involved in a variety of biological activities, through its
binding to two distinct cell surface receptors, TNFR-1 and TNFR-2
(Tartaglia and Goeddel, Immunol. Today 13: 151-153, 1992; Rothe, et
al., Immunol. Rev. 11: 81-90, 1992; Baker and Reddy, Oncogene 17:
3261-3270, 1998). Both TNF receptors are part of the larger TNF
receptor superfamily (Smith, et al., Cell 76: 959-62, 1994; Grell,
J Inflamm. 47: 8-17, 1995), which includes CD27, CD30, CD40 and Fas
antigen, among others. These receptors share no obvious sequence
similarities in the cytoplasmic domain, with the exception of
TNFR-1 and Fas, which each have an .about.80 amino acid `death
domain` (DD) at the C-terminal with .about.28% sequence identity.
These death domains can induce apoptosis by mediating self
association of both TNFR-1 and Fas upon ligand binding to each
receptor, a critical event to trigger downstream signaling pathways
by recruiting and activating receptor-associated effector molecules
(Boldin, et al., J Biol Chem 270: 7795-7798, 1995; Tartaglia, et
al., Immunol. Today 13: 151-153, 1992). Recently, many of these
downstream signaling proteins were identified and shown to contain
a DD, which mediates the interaction with the receptor through a
DD-DD interaction. For example, the DD of TRADD (TNFR-1 associated
death domain protein) (Hsu, et al., Cell 81: 495-504, 1995), and
MADD (Schievella, et al., J Biol Chem 272: 12069-75, 1997) have
been shown to interact with TNFR1; FADD (Boldin, et al., J Biol
Chem 270: 7795-7798, 1995; Chinnaiyan, et al., Cell 81: 505-512,
1995) and RIP (Stanger, et al., Cell 81: 513-523, 1995) have been
shown to interact with FAS.
[0004] TRADD, one of the earlier TNFR-1 adapter proteins identified
(Hsu, et al., Cell 81: 495-504, 1995), is a 34 kD protein that is
recruited to the TNFR1 in a TNF dependent manner. TRADD contains
two functionally separate domains, which allow the protein to
couple to at least two distinct signaling pathways (Hsu, et al.,
Cell 84: 299-308, 1996). The C-terminal region of the protein
(aal196-301) contains a death domain that mediates the interaction
between TRADD and the death domains of TNFR1, FADD and RIP. The
recruitment of FADD initiates the activation of the caspase
cascade, which eventually leads to apoptosis. The N-terminal region
of TRADD (N-TRADD) spanning from residues 1-169 appears to be a
novel domain since a BLAST (Altschul, et al., Nucl. Acids. Res. 25:
3389-3402, 1997) search did not identify any sequence homology to
known proteins. N-TRADD is responsible for the binding of TRAF2, a
TNFR-associated factor (Hsu, et al., Cell 84: 299-308, 1996). This
interaction is mediated through the TRAF domain located in the
C-terminal region of TRAF2 (348-501), termed C-TRAF2. The
interaction of N-TRADD with C-TRAF2 initiates TRAF2 mediated
signaling processes central to the cellular inflammatory response,
such as JNK and NF-.kappa.B activation (Reinhard, et al., EMBO J.
16: 1080-1092, 1997; Song, et al., Proc. Natl. Acad. Sci. 94:
9792-9796, 1997; Rothe, et al., Immunol. Rev. 11: 81-90, 1995; Cao,
et al., Nature 383: 443-446, 1996). This crucial role of N-TRADD in
TNF signaling is supported by the observation that the expression
of N-TRADD (aa1-194) can inhibit TNF-mediated NF-.kappa.B and JNK
activation in a dominant negative manner (Kieser, et al., EMBO J.
18: 2511-2521, 1999).
[0005] In addition to the TNFR-1 pathway, TRADD is also involved in
LMP1 (Epstein-Barr virus latent membrane protein 1) mediated
pathogenesis. LMP1 is a transforming viral oncogene product that
recruits both TRADD and TRAF2 to exert its biological activities in
the cell, which include activation of NF-.kappa.B, JNK and AP1
(Yuan, Curr. Opin. Cell Biol. 9: 247-251, 1997; Farrell, Trends
Microbio. 3: 105-109, 1998). Two domains in the C-terminus of LMP1
initiate the signaling processes. The CTAR1 domain binds to TRAF2,
and the CTAR2 domain binds to TRADD. Although there are
similarities between TNFR-1 and LMP1 in their adapter proteins, the
signaling mechanisms differ. In LMP1 it is the N-TRADD region
(Kieser, et al., EMBO J. 18: 2511-2521, 1999) that interacts with
LMP1, instead of the DD region as it occurs with TNFR-1 (Hsu, Cell
81: 495-504, 1995). Also, whereas a dominant negative mutant of
TRADD (1-194) can block both NF-.kappa.B and JNK signaling in the
TNFR-1 pathway, only NF-.kappa.B activity is blocked by N-TRADD
(1-194) in the LMP-1 signaling pathway (Kieser, et al., EMBO J. 18:
2511-2521, 1999).
[0006] Recently, structures of the DD of Fas (Huang, et al., Nature
384: 638-41, 1996), p75 (Liepinsh, et al., EMBO J. 16: 4999-5005,
1997) and FADD (Jeong, et al., J Biol Chem 274: 16337-42, 1999)
have been solved, providing insight into the mechanisms by which
they regulate apoptosis. In order to understand how N-TRADD may
interact with the adapter protein TRAF2, the inventors have
determined the three dimensional structure of N-TRADD (1-169) by
NMR spectroscopy. The solution structure of N-TRADD consists of 5
alpha helices and four beta strands, arranged in a unique fashion.
Using the structure, together with site-directed mutagenesis, a
region of N-TRADD has been identified that interacts with C-TRAF2.
This information, in addition to the recently published structures
of C-TRAF2 (Park, et al., Nature 398: 533-538, 1999; PDB Accession
Nos. 1CA4 and 1CA9; McWhirter, et al., Proc. Natl. Acad. Sci. USA
96: 8408-8413, 1999; PDB Accession No. 1QSC), provides insight into
the interaction of N-TRADD and C-TRAF2, which is critical for the
design and selection of potent and selective inhibitors of TNF
signaling pathways.
SUMMARY OF THE INVENTION
[0007] The present invention relates to the three dimensional
structure of the N-terminal domain of TNFR-1 associated death
domain protein ("N-TRADD"), and more specifically, to the solution
structure of N-TRADD, as determined using spectroscopy and various
computer modeling techniques.
[0008] Particularly, the invention is further directed to the
identification, characterization and three dimensional structure of
an active site of N-TRADD that provides an attractive target for
the rational design of potent and selective inhibitors of TNF
signaling pathways.
[0009] Accordingly, the present invention discloses a solution
comprising an N-terminal domain of TNFR-1 associated death domain
protein ("N-TRADD"). The three dimensional solution structure of
N-TRADD is provided by the relative atomic structural coordinates
of FIG. 2, as obtained from spectroscopy data.
[0010] Also provided by the present invention is an active site of
a C-TRAF2 binding protein or peptide, preferably of N-TRADD,
wherein said active site is characterized by a three dimensional
structure comprising the relative structural coordinates of amino
acid residues Y16, F18, and H65 according to FIG. 2, .+-.a root
mean square deviation from the conserved backbone atoms of said
amino acids of not more than 1.5 .ANG.. Also provided for by the
present invention is an N-TRADD binding active site of C-TRAF2,
wherein said active site is characterized by a three dimensional
structure comprising the relative structural coordinates of amino
acid residues R393, Y395, D399, G400, F410, F447, R448, P449, D450,
S453, S454, S455, I465, A466, S467, G468, and P470 according to the
atomic coordinates specified in Accession Nos. 1CA4, 1CA9 and/or
1QSC of the Protein Data Bank, .+-.a root mean square deviation
from the conserved backbone atoms of said amino acids of not more
than 1.5 .ANG..
[0011] The solution coordinates of N-TRADD, an N-TRADD complex or
an N-TRADD analogue (or, in each case, portions thereof, such as a
C-TRAF2 binding site of the N-TRADD molecule, complex or analogue)
as provided by this invention may be stored in a machine-readable
form on a machine-readable storage medium, e.g. a computer hard
drive, diskette, DAT tape, etc., for display as a three-dimensional
shape or for other uses involving computer-assisted manipulation
of, or computation based on, the structural coordinates or the
three-dimensional structures they define. By way of example, the
data defining the three dimensional structure of N-TRADD, an
N-TRADD complex or of an N-TRADD analogue, or a portion thereof,
may be stored in a machine-readable storage medium, and may be
displayed as a graphical three-dimensional representation of the
relevant structural coordinates, typically using a computer capable
of reading the data from said storage medium and programmed with
instructions for creating the representation from such data.
[0012] Accordingly, the present invention provides a machine, such
as a computer, programmed in memory with the coordinates of
N-TRADD, an N-TRADD analogue or a molecule or molecular complex
comprising N-TRADD or an N-TRADD analogue, or portions thereof,
together with a program capable of converting the coordinates into
a three dimensional graphical representation of the structural
coordinates on a display connected to the machine. A machine having
a memory containing such data aids in the rational design or
selection of inhibitors of N-TRADD binding or activity, including
the evaluation of the ability of a particular chemical entity to
favorably associate with N-TRADD or with an N-TRADD complex as
disclosed herein, as well as in the modeling of compounds,
proteins, complexes, etc. related by structural or sequence
homology to N-TRADD.
[0013] The present invention is additionally directed to a method
of determining the three dimensional structure of a molecule or
molecular complex whose structure is unknown, comprising the steps
of first obtaining crystals or a solution of the molecule or
molecular complex whose structure is unknown, and then generating
X-ray diffraction data from the crystallized molecule or molecular
complex and/or generating NMR data from the solution of the
molecule or molecular complex. The generated diffraction or
spectroscopy data from the molecule or molecular complex can then
be compared with the known solution coordinates or three
dimensional structure of N-TRADD as disclosed herein, and the three
dimensional structure of the unknown molecule or molecular complex
conformed to the known N-TRADD structure using standard techniques
such as molecular replacement analysis, 2D, 3D and 4D isotope
filtering, editing and triple resonance NMR techniques, and
computer homology modeling. Alternatively, a three dimensional
model of the unknown molecule may be generated by generating a
sequence alignment between N-TRADD and the unknown molecule, based
on any or all of amino acid sequence identity, secondary structure
elements or tertiary folds, and then generating by computer
modeling a three dimensional structure for the molecule using the
three dimensional structure of, and sequence alignment with,
N-TRADD.
[0014] The present invention further provides a method for
identifying an agent that interacts with N-TRADD, comprising the
steps of determining an active site of N-TRADD using the three
dimensional N-TRADD structure, and then performing computer fitting
analyses to identify an agent which interacts with the identified
active site. Also provided is a method for identifying an agent
which is a potential inhibitor of N-TRADD binding to C-TRAF2,
comprising the steps of determining an N-TRADD binding active site
of C-TRAF2 using a three dimensional structure of C-TRAF2 according
to the atomic coordinates specified in Accession Nos. 1QSC, 1CA4
and/or 1CA9 of the Protein Data Bank, selecting or designing a
candidate inhibitor of N-TRADD binding to C-TRAF2 by performing
computer fitting analyses with the three dimensional structure of
C-TRAF2, and obtaining or synthesizing the candidate inhibitor. The
inhibitor may be selected by screening an appropriate database, may
designed de novo by analyzing the steric configurations and charge
potentials of an empty C-TRAF2 active site in conjunction with the
appropriate software programs, or may be designed using
characteristics of known inhibitors of N-TRADD binding to C-TRAF2
in order to create "hybrid" inhibitors.
[0015] Still further provided is a method for identifying a
potential inhibitor of N-TRADD, comprising the steps of using a
three dimensional structure of N-TRADD as defined by the relative
structural coordinates of amino acids encoding N-TRADD to design or
select a potential inhibitor, and obtaining or synthesizing said
potential inhibitor. The inhibitor may be selected by screening an
appropriate database, may designed de novo by analyzing the steric
configurations and charge potentials of an empty N-TRADD active
site in conjunction with the appropriate software programs, or may
be designed using characteristics of known inhibitors of N-TRADD,
an N-TRADD complex or of an N-TRADD analogue in order to create
"hybrid" inhibitors. Also provided by the present invention are the
inhibitors designed or selected using the methods disclosed
herein.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1 depicts the 169 amino acid sequence encoding the
N-terminal domain of human TNFR-1 associated death domain protein
(wherein said N-terminal domain is referred to herein as
"N-TRADD"), with the secondary structures noted below.
[0017] FIG. 2 lists the atomic structure coordinates for the
restrained minimized mean structure of N-TRADD as derived by
multidimensional NMR spectroscopy. "Atom type" refers to the atom
whose coordinates are being measured. "Residue" refers to the type
of residue of which each measured atom is a part--i.e., amino acid,
cofactor, ligand or solvent. The "x, y and z" coordinates indicate
the Cartesian coordinates of each measured atom's location (.ANG.).
All non-protein atoms are listed as HETATM instead of atoms using
PDB conventions.
[0018] FIG. 3 depicts the relative binding affinities of wild type
N-TRADD and five N-TRADD mutants to C-TRAF2. Each experiment was
performed three times, and the standard deviation is shown as thin
lines above the bars.
DETAILED DESCRIPTION OF THE INVENTION
[0019] As used herein, the following terms and phrases shall have
the meanings set forth below:
[0020] Unless otherwise noted, "N-TRADD" includes both the
N-terminal domain of TRADD as encoded by the amino acid sequence of
FIG. 1 (including conservative substitutions thereof), as well as
"N-TRADD analogues", defined herein as proteins comprising a
C-TRAF2 or C-TRAF2 like binding active site as defined by the
present invention, including, but not limited to, an active site
characterized by a three dimensional structure comprising the
relative structural coordinates of amino acid residues Y16, F18,
and H65 according to FIG. 2, or more preferably, further comprising
the relative structural coordinates of amino acid residues L17,
V58, L59, 172, and D149 according to FIG. 2, or even more
preferably, still further comprising the relative structural
coordinates of amino acid residues K63, I64, D68, Q70, V73, Q74,
L75, C78, L118, G121, A122, R124, L125, E150, and L152 according to
FIG. 2, in each case, .+-.a root mean square deviation from the
conserved backbone atoms (N, C.alpha., C, and O) of said amino
acids of not more than 1.5 .ANG., or more preferably, not more than
1.0 .ANG., or most preferably, not more than 0.5 .ANG..
[0021] "N-TRADD function or activity" shall include LMP1 mediated
pathogenesis and a variety of TNF signaling processes, such as
TRAF2 mediated signaling processes central to the cellular
inflammatory response, including, but not limited to, JNK and
NF-.kappa.B activation.
[0022] Unless otherwise indicated, "protein" or "molecule" shall
include a protein, protein domain, polypeptide or peptide.
[0023] "Structural coordinates" are the Cartesian coordinates
corresponding to an atom's spatial relationship to other atoms in a
molecule or molecular complex. Structural coordinates may be
obtained using x-ray crystallography techniques or NMR techniques,
or may be derived using molecular replacement analysis or homology
modeling. Various software programs allow for the graphical
representation of a set of structural coordinates to obtain a three
dimensional representation of a molecule or molecular complex. The
structural coordinates of the present invention may be modified
from the original set provided in FIG. 2 by mathematical
manipulation, such as by inversion or integer additions or
subtractions. As such, it is recognized that the structural
coordinates of the present invention are relative, and are in no
way specifically limited by the actual x, y, z coordinates of FIG.
2.
[0024] An "agent" shall include a protein, polypeptide, peptide,
nucleic acid, including DNA or RNA, molecule, compound, antibiotic
or drug.
[0025] "Root mean square deviation" is the square root of the
arithmetic mean of the squares of the deviations from the mean, and
is a way of expressing deviation or variation from the structural
coordinates described herein. The present invention includes all
embodiments comprising conservative substitutions of the noted
amino acid residues resulting in same structural coordinates within
the stated root mean square deviation.
[0026] It will be obvious to the skilled practitioner that the
numbering of the amino acid residues in the various isoforms of
N-TRADD or in N-TRADD analogues covered by the present invention
may be different than that set forth herein, or may contain certain
conservative amino acid substitutions that yield the same three
dimensional or solution structures as those defined by FIG. 2
herein. Corresponding amino acids and conservative substitutions in
other isoforms or analogues are easily identified by visual
inspection of the relevant amino acid sequences or by using
commercially available homology software programs.
[0027] "Conservative substitutions" are those amino acid
substitutions which are functionally equivalent to the substituted
amino acid residue, either by way of having similar polarity,
steric arrangement, or by belonging to the same class as the
substituted residue (e.g., hydrophobic, acidic or basic), and
includes substitutions having an inconsequential effect on the
three dimensional structure of N-TRADD with respect to the use of
said structure for the identification and design of N-TRADD or
N-TRADD complex inhibitors, for molecular replacement analyses
and/or for homology modeling.
[0028] An "active site" refers to a region of a molecule or
molecular complex that, as a result of its shape and charge
potential, favorably interacts or associates with another agent
(including, without limitation, a protein, polypeptide, peptide,
nucleic acid, including DNA or RNA, molecule, compound, antibiotic
or drug) via various covalent and/or non-covalent binding forces.
As such, an active site of the present invention may include both
the actual site of C-TRAF2 binding with N-TRADD, as well as
accessory binding sites adjacent to the actual site of C-TRAF2
binding that nonetheless may affect N-TRADD or N-TRADD complex
activity upon interaction or association with a particular agent,
either by direct interference with the actual site of C-TRAF2
binding or by indirectly affecting the steric conformation or
charge potential of the N-TRADD molecule and thereby preventing or
reducing C-TRAF2 binding to N-TRADD at the actual site of C-TRAF2
binding.
[0029] An "N-TRADD complex" refers to a co-complex of a molecule
comprising the N-TRADD region in bound association with a protein,
polypeptide, peptide, nucleic acid, including DNA or RNA, small
molecule, compound, antibiotic or drug, either by covalent or
non-covalent binding forces. A non-limiting example of an N-TRADD
complex includes N-TRADD or an N-TRADD analogue bound to
C-TRAF2.
[0030] The present invention relates to the three dimensional
structure of N-TRADD or of an N-TRADD analogue, and more
specifically, to the solution structure of N-TRADD as determined
using multidimensional NMR spectroscopy and various computer
modeling techniques. The solution coordinates of N-TRADD (disclosed
herein at FIG. 2) are useful for a number of applications,
including, but not limited to, the characterization of a three
dimensional structure of N-TRADD, as well as the visualization,
identification and characterization of N-TRADD active sites,
including the site of C-TRAF2 binding to N-TRADD. The active site
structures may then be used to predict the orientation and binding
affinity of a designed or selected inhibitor of N-TRADD, an N-TRADD
analogue or of an N-TRADD complex. Accordingly, the invention is
particularly directed to the three dimensional structure of an
N-TRADD active site, including but not limited to the C-TRAF2
binding site.
[0031] As used herein, N-TRADD comprises the N-terminal domain of
TRADD, and more specifically comprises amino acid residues 1-169 of
TRADD as shown in FIG. 1 ("N-TRADD"), or conservative substitutions
thereof. The present invention provides a solution comprising an
N-terminal domain of TNFR-1 associated death domain protein,
wherein the N-terminal domain of TNFR-1 associated death domain
protein preferably comprises amino acid residues 1-169 of FIG. 1,
or conservative substitutions thereof. Preferably, the solution
provided for herein comprises N-TRADD in a buffer comprising 20 mM
imidazole, 200 mM NaCl, 20 mM DTT and 0.05% NaN.sub.3, in either
90% H.sub.2O/10% D.sub.2O or 100% D.sub.2O. The concentration of
protein in the solution should be high enough to yield a good
signal-to-noise ratio in the NMR spectrum, but not so high as to
result in precipitation or aggregation of the protein or protein
complex. By way of example, the solutions of the present invention
preferably comprise between 0.8-1.0 mM uncomplexed N-TRADD.
However, it is understood that one of ordinary skill in the art may
devise additional solutions using alternate molar concentrations
that are still able to obtain a usable NMR spectrum. A preferred
solution pH is around 6.6. Further, the N-TRADD of the solutions of
the present invention may be either unlabeled, .sup.15N enriched or
.sup.15N,.sup.13C enriched, and is preferably biologically
active.
[0032] The secondary structure of the N-TRADD used in the solutions
of the present invention comprises four beta strands forming an
antiparallel beta sheet, with five alpha helices packing around the
beta sheet, wherein the beta strands and alpha helices are
configured in the trace order .beta.1, .alpha.1, .alpha.2, .beta.2,
.beta.3, .alpha.3, .beta.4, .alpha.4 and .alpha.5. The .beta.1
strand comprises amino acid residues S14-E20 of N-TRADD, .alpha.1
comprises amino acid residues L28-Y32 of N-TRADD, .alpha.2
comprises amino acid residues P35-G53 of N-TRADD, .beta.2 comprises
amino acid residues Q60-R66 of N-TRADD, .beta.3 comprises amino
acid residues L71-R76 of N-TRADD, .alpha.3 comprises amino acid
residues R80-L107 of N-TRADD, .beta.4 comprises amino acid residues
Q115-R119 of N-TRADD, .alpha.4 comprises amino acid residues
E132-A141 of N-TRADD and .alpha.5 comprises amino acid residues
E150-N161 of N-TRADD.
[0033] The protein used in the solutions of the present invention
includes N-TRADD, as well as N-TRADD analogues, where said protein
comprises an active site characterized by the three dimensional
structure comprising the relative structural coordinates of amino
acid residues Y16, F18, and H65 according to FIG. 2, .+-.a root
mean square deviation from the conserved backbone atoms of said
amino acids of not more than 1.5 .ANG., or preferably, not more
than 1.0 .ANG., or more preferably not more than 0.5 .ANG.. In a
preferred embodiment, the protein used in the solutions of the
present invention comprises an active site characterized by a three
dimensional structure further comprising the relative structural
coordinates of amino acid residues L17, V58, L59, 172, and D149
according to FIG. 2, .+-.a root mean square deviation from the
conserved backbone atoms of said amino acids of not more than 1.5
.ANG., or preferably, not more than 1.0 .ANG., or most preferably,
not more than 0.5 .ANG.. In still more preferred embodiment, the
protein used in the solutions of the present invention comprises an
active site characterized by the three dimensional structure still
further comprising the relative structural coordinates of amino
acid residues K63, I64, D68, Q70, V73, Q74, L75, C78, L118, G121,
A122, R124, L125, E150, and L152 according to FIG. 2, .+-.a root
mean square deviation from the conserved backbone atoms of said
amino acids of not more than 1.5 .ANG., or preferably, not more
than 1.0 .ANG., or most preferably not more than 0.5 .ANG.. In the
most preferred embodiment, the protein used in the solution of the
present invention is characterized by a three dimensional structure
comprising the complete structural coordinates of the amino acids
according to FIG. 2, .+-.a root mean square deviation from the
conserved backbone atoms of said amino acids of not more than 1.5
.ANG. (or more preferably, not more than 1.0 .ANG., and most
preferably, not more than 0.5 .ANG.).
[0034] Molecular modeling methods known in the art may be used to
identify an active site or binding pocket of N-TRADD, an N-TRADD
molecular complex, or of an N-TRADD analogue. Specifically, the
solution structural coordinates provided by the present invention
may be used to characterize a three dimensional structure of the
N-TRADD molecule, molecular complex or N-TRADD analogue. From such
a structure, putative active sites may be computationally
visualized, identified and characterized based on the surface
structure of the molecule, surface charge, steric arrangement, the
presence of reactive amino acids, regions of hydrophobicity or
hydrophilicity, etc. Such putative active sites may be further
refined using chemical shift perturbations of spectra generated
from various and distinct N-TRADD complexes, competitive and
non-competitive inhibition experiments, and/or by the generation
and characterization of N-TRADD or ligand mutants to identify
critical residues or characteristics of the active site.
[0035] The identification of putative active sites of a molecule or
molecular complex is of great importance, as most often the
biological activity of a molecule or molecular complex results from
the interaction between an agent and one or more active sites of
the molecule or molecular complex. Accordingly, the active sites of
a molecule or molecular complex are the best targets to use in the
design or selection of inhibitors that affect the activity of the
molecule or molecular complex.
[0036] The present invention is directed to an active site of
N-TRADD, an N-TRADD complex or of an N-TRADD analogue, that, as a
result of its shape, reactivity, charge potential, etc., favorably
interacts or associates with another agent (including, without
limitation, a protein, polypeptide, peptide, nucleic acid,
including DNA or RNA, molecule, compound, antibiotic or drug).
Accordingly, the present invention is directed to an active site of
the N-TRADD molecule characterized by the three dimensional
structure comprising the relative structural coordinates of amino
acid residues Y16, F18, and H65 according to FIG. 2, .+-.a root
mean square deviation from the conserved backbone atoms of said
amino acids of not more than 1.5 .ANG., or preferably, not more
than 1.0 .ANG., or more preferably not more than 0.5 .ANG..
Preferably, the active site of the N-TRADD molecule is
characterized by the three dimensional structure further comprising
the relative structural coordinates of amino acid residues L17,
V58, L59, I72, and D149, .+-.a root mean square deviation from the
conserved backbone atoms of said amino acids of not more than 1.5
.ANG., or preferably, not more than 1.0 .ANG., or more preferably
not more than 0.5 .ANG.. Most preferably, an active site of the
N-TRADD molecule is characterized by the three dimensional
structure still further comprising the relative structural
coordinates of amino acid residues K63, I64, D68, Q70, V73, Q74,
L75, C78, L118, G121, A122, R124, L125, E150, and L152 according to
FIG. 2, .+-.a root mean square deviation from the conserved
backbone atoms of said amino acids of not more than 1.5 .ANG., or
preferably, not more than 1.0 .ANG., or more preferably not more
than 0.5 .ANG..
[0037] In order to use the structural coordinates generated for a
solution structure of the present invention as set forth in FIG. 2,
it is often necessary to display the relevant coordinates as, or
convert them to, a three dimensional shape or graphical
representation, or to otherwise manipulate them. For example, a
three dimensional representation of the structural coordinates is
often used in rational drug design, molecular replacement analysis,
homology modeling, and mutation analysis. This is typically
accomplished using any of a wide variety of commercially available
software programs capable of generating three dimensional graphical
representations of molecules or portions thereof from a set of
structural coordinates. Examples of said commercially available
software programs include, without limitation, the following: GRID
(Oxford University, Oxford, UK); MCSS (Molecular Simulations, San
Diego, Calif.); AUTODOCK (Scripps Research Institute, La Jolla,
Calif.); DOCK (University of California, San Francisco, Calif.);
Flo99 (Thistlesoft, Morris Township, N.J.); Ludi (Molecular
Simulations, San Diego, Calif.); QUANTA (Molecular Simulations, San
Diego, Calif.); Insight (Molecular Simulations, San Diego, Calif.);
SYBYL (TRIPOS, Inc., St. Louis. Mo.); and LEAPFROG (TRIPOS, Inc.,
St. Louis, Mo.).
[0038] For storage, transfer and use with such programs, a machine,
such as a computer, is provided for that produces a three
dimensional representation of the N-TRADD molecule, a portion
thereof (such as an active site or a binding site), an N-TRADD
molecular complex, or an N-TRADD analogue. The machine of the
present invention comprises a machine-readable data storage medium
comprising a data storage material encoded with machine-readable
data. Machine-readable storage media comprising data storage
material include conventional computer hard drives, floppy disks,
DAT tape, CD-ROM, and other magnetic, magneto-optical, optical,
floptical and other media which may be adapted for use with a
computer. The machine of the present invention also comprises a
working memory for storing instructions for processing the
machine-readable data, as well as a central processing unit (CPU)
coupled to the working memory and to the machine-readable data
storage medium for the purpose of processing the machine-readable
data into the desired three dimensional representation. Finally,
the machine of the present invention further comprises a display
connected to the CPU so that the three dimensional representation
may be visualized by the user. Accordingly, when used with a
machine programmed with instructions for using said data, e.g., a
computer loaded with one or more programs of the sort identified
above, the machine provided for herein is capable of displaying a
graphical three-dimensional representation of any of the molecules
or molecular complexes, or portions of molecules of molecular
complexes, described herein.
[0039] In one embodiment of the invention, the machine-readable
data comprises the relative structural coordinates of amino acid
residues Y16, F18, and H65 according to FIG. 2, .+-.a root mean
square deviation from the conserved backbone atoms of said amino
acids of not more than 1.5 .ANG., or preferably, not more than 1.0
.ANG., or more preferably not more than 0.5 .ANG.. In an alternate
preferred embodiment, the machine-readable data further comprises
the relative structural coordinates of amino acid residues L17,
V58, L59, I72, and D149 according to FIG. 2, .+-.a root mean square
deviation from the conserved backbone atoms of said amino acids of
not more than 1.5 .ANG., or preferably, not more than 1.0 .ANG., or
more preferably not more than 0.5 .ANG.. In a still more preferred
embodiment, the machine-readable data still further comprises the
relative structural coordinates of amino acid residues K63, I64,
D68, Q70, V73, Q74, L75, C78, L118, G121, A122, R124, L125, E150,
and L152 according to FIG. 2, .+-.a root mean square deviation from
the conserved backbone atoms of said amino acids of not more than
1.5 .ANG., or preferably, not more than 1.0 .ANG., or more
preferably not more than 0.5 .ANG.. Finally, in the most preferred
embodiment, the machine readable data comprises the complete
structural coordinates according to FIG. 2, .+-.a root mean square
deviation of not more than 1.5 .ANG. (or more preferably, not more
than 1.0 .ANG., and most preferably, not more than 0.5 .ANG.).
[0040] The structural coordinates of the present invention permit
the use of various molecular design and analysis techniques in
order to (i) solve the three dimensional structures of related
molecules, molecular complexes or N-TRADD analogues, and (ii) to
design, select, and synthesize chemical agents capable of favorably
associating or interacting with an active site of an N-TRADD
molecule, molecular complex or N-TRADD analogue, wherein said
chemical agents potentially act as inhibitors of N-TRADD or N-TRADD
complex binding to a number of binding proteins, including, but not
limited to, C-TRAF2.
[0041] More specifically, the present invention provides a method
for determining the molecular structure of a molecule or molecular
complex whose structure is unknown, comprising the steps of
obtaining a solution of the molecule or molecular complex whose
structure is unknown, and then generating NMR data from the
solution of the molecule or molecular complex. The NMR data from
the molecule or molecular complex whose structure is unknown is
then compared to the solution structure data obtained from the
N-TRADD solutions of the present invention. Then, 2D, 3D and 4D
isotope filtering, editing and triple resonance NMR techniques are
used to conform the three dimensional structure determined from the
N-TRADD solution of the present invention to the NMR data from the
solution molecule or molecular complex. Alternatively, molecular
replacement analysis may be used to conform the N-TRADD solution
structure of the present invention to x-ray diffraction data from
crystals of the unknown molecule or molecular complex.
[0042] Molecular replacement analysis uses a molecule having a
known structure as a starting point to model the structure of an
unknown crystalline sample. This technique is based on the
principle that two molecules which have similar structures,
orientations and positions will diffract x-rays similarly. A
corresponding approach to molecular replacement is applicable to
modeling an unknown solution structure using NMR technology. The
NMR spectra and resulting analysis of the NMR data for two similar
structures will be essentially identical for regions of the
proteins that are structurally conserved, where the NMR analysis
consists of obtaining the NMR resonance assignments and the
structural constraint assignments, which may contain hydrogen bond,
distance, dihedral angle, coupling constant, chemical shift and
dipolar coupling constant constraints. The observed differences in
the NMR spectra of the two structures will highlight the
differences between the two structures and identify the
corresponding differences in the structural constraints. The
structure determination process for the unknown structure is then
based on modifying the NMR constraints from the known structure to
be consistent with the observed spectral differences between the
NMR spectra.
[0043] Accordingly, in one non-limiting embodiment of the
invention, the resonance assignments for the N-TRADD solution
provide the starting point for resonance assignments of N-TRADD in
a new N-TRADD:"unsolved agent" complex. Chemical shift perturbances
in two dimensional .sup.15N/.sup.1H spectra can be observed and
compared between the N-TRADD solution and the new N-TRADD:agent
complex. In this way, the affected residues may be correlated with
the three dimensional structure of N-TRADD as provided by the
relevant structural coordinates of FIG. 2. This effectively
identifies the region of the N-TRADD:agent complex that has
incurred a structural change relative to the native N-TRADD
structure. The .sup.1H, .sup.15N, .sup.13C and .sup.13CO NMR
resonance assignments corresponding to both the sequential backbone
and side-chain amino acid assignments of N-TRADD may then be
obtained and the three dimensional structure of the new
N-TRADD:agent complex may be generated using standard 2D, 3D and 4D
triple resonance NMR techniques and NMR assignment methodology,
using the N-TRADD solution structure, resonance assignments and
structural constraints as a reference. Various computer fitting
analyses of the new agent with the three dimensional model of
N-TRADD may be performed in order to generate an initial three
dimensional model of the new agent complexed with N-TRADD, and the
resulting three dimensional model may be refined using standard
experimental constraints and energy minimization techniques in
order to position and orient the new agent in association with the
three dimensional structure of N-TRADD.
[0044] The present invention further provides that the structural
coordinates of the present invention may be used with standard
homology modeling techniques in order to determine the unknown
three-dimensional structure of a molecule or molecular complex.
Homology modeling involves constructing a model of an unknown
structure using structural coordinates of one or more related
protein molecules, molecular complexes or parts thereof (i.e.,
active sites). Homology modeling may be conducted by fitting common
or homologous portions of the protein whose three dimensional
structure is to be solved to the three dimensional structure of
homologous structural elements in the known molecule, specifically
using the relevant (i.e., homologous) structural coordinates
provided by FIG. 2 herein. Homology may be determined using amino
acid sequence identity, homologous secondary structure elements,
and/or homologous tertiary folds. Homology modeling can include
rebuilding part or all of a three dimensional structure with
replacement of amino acids (or other components) by those of the
related structure to be solved.
[0045] Accordingly, a three dimensional structure for the unknown
molecule or molecular complex may be generated using the three
dimensional structure of the N-TRADD molecule of the present
invention, refined using a number of techniques well known in the
art, and then used in the same fashion as the structural
coordinates of the present invention, for instance, in applications
involving molecular replacement analysis, homology modeling, and
rational drug design.
[0046] Determination of the three dimensional structure of N-TRADD
and its C-TRAF2 binding active site as disclosed herein is critical
to the rational identification and/or design of agents that may act
as inhibitors of C-TRAF2 binding to N-TRADD, and thereby act as
inhibitors of JNK/AP1 and NF-.kappa.B activation. Alternatively,
using conventional drug assay techniques, the only way to identify
such an agent is to screen thousands of test compounds until an
agent having the desired inhibitory effect on a target compound is
identified. Necessarily, such conventional screening methods are
expensive, time consuming, and do not elucidate the method of
action of the identified agent on the target compound.
[0047] However, advancing X-ray, spectroscopic and computer
modeling technologies allow researchers to visualize the three
dimensional structure of a targeted compound (i.e., of N-TRADD).
Using such a three dimensional structure, researchers identify
putative binding sites and then identify or design agents to
interact with these binding sites. These agents are then screened
for an inhibitory effect upon the target molecule. In this manner,
not only are the number of agents to be screened for the desired
activity greatly reduced, but the mechanism of action on the target
compound is better understood.
[0048] Accordingly, the present invention further provides a method
for identifying a potential inhibitor of N-TRADD, an N-TRADD
analogue or of an N-TRADD complex, comprising the steps of using a
three dimensional structure of N-TRADD as defined by the relative
structural coordinates of FIG. 2 to design or select a potential
inhibitor of N-TRADD activity, and synthesizing or obtaining said
potential inhibitor. The inhibitor may be selected by screening an
appropriate database, may be designed de novo by analyzing the
steric configurations and charge potentials of an empty N-TRADD or
N-TRADD complex active site in conjunction with the appropriate
software programs, or may be designed using characteristics of
known inhibitors of protein binding to N-TRADD or N-TRADD complexes
in order to create "hybrid" inhibitors.
[0049] An agent that interacts or associates with an active site of
N-TRADD, an N-TRADD complex or an N-TRADD analogue may be
identified by determining an active site from the three dimensional
structure of N-TRADD, and performing computer fitting analyses to
identify an agent which interacts or associates with said active
site. Computer fitting analyses utilize various computer software
programs that evaluate the "fit" between the putative active site
and the identified agent, by (a) generating a three dimensional
model of the putative active site of a molecule or molecular
complex using homology modeling or the atomic structural
coordinates of the active site, and (b) determining the degree of
association between the putative active site and the identified
agent. The degree of association may be determined computationally
by any number of commercially available software programs, or may
be determined experimentally using standard binding assays.
[0050] Three dimensional models of the putative active site may be
generated using any one of a number of methods known in the art,
and include, but are not limited to, homology modeling as well as
computer analysis of raw structural coordinate data generated using
crystallographic or spectroscopy techniques. Computer programs used
to generate such three dimensional models and/or perform the
necessary fitting analyses include, but are not limited to: GRID
(Oxford University, Oxford, UK), MCSS (Molecular Simulations, San
Diego, Calif.), AUTODOCK (Scripps Research Institute, La Jolla,
Calif.), DOCK (University of California, San Francisco, Calif.),
Flo99 (Thistlesoft, Morris Township, N.J.), Ludi (Molecular
Simulations, San Diego, Calif.), QUANTA (Molecular Simulations, San
Diego, Calif.), Insight (Molecular Simulations, San Diego, Calif.),
SYBYL (TRIPOS, Inc., St. Louis. Mo.) and LEAPFROG (TRIPOS, Inc.,
St. Louis, Mo.).
[0051] In a preferred method of the present invention, the
identified active site of N-TRADD, an N-TRADD complex or of an
N-TRADD analogue comprises amino acid residues Y16, F18 and H65 (or
conservative substitutions thereof) according to FIG. 1, more
preferably further comprises amino acid residues L17, V58, L59,
I72, and D149 (or conservative substitutions thereof) according to
FIG. 1, and most preferably still further comprises amino acid
residues K63, I64, D68, Q70, V73, Q74, L75, C78, L118, G121, A122,
R124, L125, E150, and L152 (or conservative substitutions thereof)
according to FIG. 1.
[0052] The method of the present invention also comprises an
identified active site characterized by the three dimensional
structure comprising the relative structural coordinates of amino
acid residues Y16, F18, and H65 according to FIG. 2, .+-.a root
mean square deviation from the conserved backbone atoms of said
amino acids of not more than 1.5 .ANG., or preferably, not more
than 1.0 .ANG., or more preferably not more than 0.5 .ANG..
Preferably, the identified active site is characterized by three
dimensional structure further comprising the relative structural
coordinates of amino acid residues L17, V58, L59, I72, and D149
according to FIG. 2, and most preferably still further comprising
the relative structural coordinates of amino acid residues K63,
I64, D68, Q70, V73, Q74, L75, C78, L118, G121, A122, R124, L125,
E150, and L152, in each case, .+-.a root mean square deviation from
the conserved backbone atoms of said amino acids of not more than
1.5 .ANG., or preferably, not more than 1.0 .ANG., or more
preferably not more than 0.5 .ANG.. It is understood that the
method of the present invention includes additional embodiments
comprising conservative substitutions of the noted amino acids
which result in the same structural coordinates of the
corresponding residues in FIG. 2 within the stated root mean square
deviation.
[0053] The effect of such an agent identified by computer fitting
analyses on N-TRADD, an N-TRADD complex or an N-TRADD analogue
activity may be further evaluated computationally, or
experimentally by competitive binding experiments or by contacting
the identified agent with N-TRADD (or an N-TRADD complex or
analogue) and measuring the effect of the agent on the target's
biological activity. Standard enzymatic assays may be performed and
the results analyzed to determine whether the agent is an inhibitor
of N-TRADD activity (i.e., the agent may reduce or prevent binding
affinity between N-TRADD and the relevant binding protein, such as
C-TRAF2, and thereby reduce the level or rate of JNK/AP1 and/or
NF-.kappa.B activity compared to baseline). Further tests may be
performed to evaluate the selectivity of the identified agent to
N-TRADD with regard to other N-TRADD analogues or C-TRAF2 binding
targets.
[0054] Agents designed or selected to interact with N-TRADD or an
N-TRADD complex must be capable of both physically and structurally
associating with N-TRADD via various covalent and/or non-covalent
molecular interactions, and of assuming a three dimensional
configuration and orientation that complements the relevant active
site of the N-TRADD molecule or of the N-TRADD complex.
[0055] Accordingly, using these criteria, the structural
coordinates of the N-TRADD molecule as disclosed herein, and/or
structural coordinates derived therefrom using molecular
replacement analysis or homology modeling, agents may be designed
to increase either or both of the potency and selectivity of known
inhibitors, either by modifying the structure of known inhibitors
or by designing new agents de novo via computational inspection of
the three dimensional configuration and electrostatic potential of
an N-TRADD or N-TRADD complex active site.
[0056] Accordingly, in one embodiment of the invention, the
structural coordinates of FIG. 2 of the present invention, or
structural coordinates derived therefrom using molecular
replacement or homology modeling techniques as discussed above, are
used to screen a database for agents that may act as potential
inhibitors of N-TRADD or N-TRADD complex activity. Specifically,
the obtained structural coordinates of the present invention are
read into a software package and the three dimensional structure is
analyzed graphically. A number of computational software packages
may be used for the analysis of structural coordinates, including,
but not limited to, Sybyl (Tripos Associates), QUANTA and XPLOR
(Brunger, A. T., (1994) X-Plor 3.851: a system for X-ray
Crystallography and NMR. Xplor Version 3.851 New Haven, Conn.: Yale
University Press). Additional software programs check for the
correctness of the coordinates with regard to features such as bond
and atom types. If necessary, the three dimensional structure is
modified and then energy minimized using the appropriate software
until all of the structural parameters are at their
equilibrium/optimal values. The energy minimized structure is
superimposed against the original structure to make sure there are
no significant deviations between the original and the energy
minimized coordinates.
[0057] The energy minimized coordinates of N-TRADD or of an N-TRADD
complex bound to a "solved" inhibitor are then analyzed and the
interactions between the solved ligand and N-TRADD or the N-TRADD
complex are identified. The final N-TRADD or N-TRADD complex
structure is modified by graphically removing the solved inhibitor
so that only N-TRADD or the N-TRADD complex and a few residues of
the solved agent are left for analysis of the binding site cavity.
QSAR and SAR analysis and/or conformational analysis may be carried
out to determine how other inhibitors compare to the solved
inhibitor. The solved agent may be docked into the uncomplexed
structure's binding site to be used as a template for data base
searching, using software to create excluded volume and distance
restrained queries for the searches. Structures qualifying as hits
are then screened for activity using standard assays and other
methods known in the art.
[0058] Further, once the specific interaction is determined between
the solved inhibitor, docking studies with different inhibitors
allow for the generation of initial models of new inhibitors bound
to N-TRADD or to the N-TRADD complex. The integrity of these new
models may be evaluated a number of ways, including constrained
conformational analysis using molecular dynamics methods (i.e.,
where both N-TRADD (or the N-TRADD complex) and the bound inhibitor
are allowed to sample different three dimensional conformational
states until the most favorable state is reached or found to exist
between the protein (or protein complex) and the bound agent). The
final structure as proposed by the molecular dynamics analysis is
analyzed visually to make sure that the model is in accord with
known experimental SAR based on measured binding affinities. Once
models are obtained of the original solved agent bound to N-TRADD
or the N-TRADD complex and computer models of other molecules bound
to N-TRADD or the N-TRADD complex, strategies are determined for
designing modifications into the inhibitors to improve their
activity and/or enhance their selectivity.
[0059] Once an N-TRADD or N-TRADD complex binding agent has been
optimally selected or designed, as described above, substitutions
may then be made in some of its atoms or side groups in order to
improve or modify its selectivity and binding properties.
Generally, initial substitutions are conservative, i.e., the
replacement group will have approximately the same size, shape,
hydrophobicity and charge as the original group. Such substituted
chemical compounds may then be analyzed for efficiency of fit to
the N-TRADD molecule or the N-TRADD complex by the same computer
methods described in detail above.
[0060] Various molecular analysis and rational drug design
techniques are further disclosed in U.S. Pat. Nos. 5,834,228,
5,939,528 and 5,865,116, as well as in PCT Application No.
PCT/US98/16879, published as WO 99/09148, the contents of which are
hereby incorporated by reference.
[0061] The present invention may be better understood by reference
to the following non-limiting Example. The following Example is
presented in order to more fully illustrate the preferred
embodiments of the invention, and should in no way be construed as
limiting the scope of the present invention.
EXAMPLE 1
[0062] The structure of N-TRADD was determined by NMR spectroscopy.
The structure consists of four .beta. strands which form an
antiparallel beta sheet, with .alpha. helices packing around the
sheet. N-TRADD interacts with the C-terminal domain of TRAF2
("C-TRAF2") to initiate one of the most important TNFR1 activities,
NF-.kappa.B and JNK activation. N-TRADD residues involved in
C-TRAF2 interaction were identified from NMR binding experiments of
N-TRADD with C-TRAF2, where several residues important for the
binding were determined to be located primarily in the antiparallel
beta sheet. The mutation of some N-TRADD residues that were
identified from .sup.1H-.sup.15N HSQC perturbations to be involved
in C-TRAF2 binding caused a 2-16 fold decrease in the affinity of
N-TRADD for C-TRAF2. Interestingly, the interaction between N-TRADD
and C-TRAF2 was inhibited by a 13-mer peptide derived from CD40,
inferring that the binding site of N-TRADD and CD40 in C-TRAF2
overlap. The knowledge of the N-TRADD NMR structure and the C-TRAF2
crystal structure (McWhirter, Proc. Natl. Acad. Sci. USA 96:
8408-8413, 1999; published PDB Accession No. 1QSC), in addition to
the characterization of their interaction sites, are critical
components in the design of drugs that may inhibit their
interactions, therefore allowing intervention of the inflammatory
cascade in the TNFR1 pathway.
[0063] Experimental Methods
[0064] Cloning and site directed mutagenesis: The DNA sequence
coding for the first 169 amino acids from N-TRADD was cloned in
pRSETB (Invitrogen) after amplification by PCR. The primer at the
5' end introduces an NdeI site upstream from the initiation site,
and the primer at the 3' end introduces a His tag after aa169. The
sequence was confirmed by sequencing analysis. Single point
mutations were introduced using Chameleon double-stranded site
directed mutagenesis (Stratagene). The C-TRAF2 sequence (residues
262 to 501 of human TRAF2) was amplified by PCR using a 5' end
primer flanking with an XhoI and NdeI site, which introduces a Met
before residue 262, and a 3' end primer introducing 6.times.His
before a stop codon followed by a NcoI site. The C-TRAF2 sequence
was cloned into pAcSG2 (Pharmingen) by XhoI and NcoI sites for
baculovirus expression.
[0065] Protein expression and purification: The polypeptide for
N-TRADD (1-169) was overexpressed in BL-21 E. coli. [U-.sup.15N] or
[U-.sup.13C, U-.sup.15N]. N-TRADD was purified from cells grown at
25.degree. C. on minimal medium containing 2 g/L [.sup.15N]ammonium
sulfate or [.sup.15N]ammonium sulfate and 2 g/L [U-.sup.13C]
glucose. The cells were lysed at 4.degree. C. in 20 mM Tris-HCl
(pH=8.0), 20 mM DTT (buffer B) with 200 mM NaCl. The lysate was
centrifuged at 21000.times.g at 4.degree. C. for 60 minutes. All
purification steps were performed at room temperature unless noted
otherwise. The supernatant containing soluble N-TRADD was diluted
four-fold with buffer B and subjected to anion exchange
chromatography using a ToyoPearl Q 550C column. The resulting
unbound fraction was applied to a ToyoPearl AF-Heparin 650M column.
The heparin unbound fraction was diluted 2-fold with buffer B and
applied to a Pharmacia Blue Sepharose CL-6B column. N-TRADD was
recovered using a linear gradient with buffer B from 0-0.5 M NaCl.
The N-TRADD containing fraction was concentrated and applied to a
ToyoPearl G3000SW.times.1 size exclusion column equilibrated with
20 mM imidazole (pH=6.6), 200 mM NaCl, 20 mM DTT, and 10% D.sub.2O.
The resulting N-TRADD sample was judged to be pure (>95%) by
SDS-PAGE and monomeric (>95%) by SEC-HPLC. All NMR samples were
in 20 mM imidazole, pH 6.6, 200 mM NaCl, 20 mM DTT and 0.05% NaN3,
at concentrations between 0.8-1.0 mM with 90% H.sub.2O/10% D.sub.2O
or 100% D.sub.2O.
[0066] C-TRAF2 protein was expressed in baculovirus in Tini cells.
The cell pellet was resuspended in the buffer A (20 mM Tris pH 7.5,
200 mM NaCl, 10% glycerol) containing 2 mM PMSF, 10 ug/ml leupeptin
and 5 ug/ml aprotinin. Cells were then lysed by sonication.
Extracts were clarified by centrifugation at 18K for 30 min and
applied to Ni-NTA agarose (Qiagen). The column was washed with
buffer A containing 20 mM imidazole and later, 50 mM imidazole.
C-TRAF2 protein was then eluted with buffer A containing 250 mM
imidazole and 10 mM DTT. The eluted protein was then diluted with
50 mM Tris pH7.5 and 10 mM DTT and applied to a Toyopearl QAE-550C
column. The unbound fraction was collected and applied to a
Toyopearl AF Heparin-650M column equilibrated with 50 mM Tris pH
7.5 and 10 mM DTT. The column was then eluted with 0-1 N NaCl
gradient. The C-TRAF2 protein was eluted at about 200 mM NaCl. The
purity of C-TRAF2 was greater than 90% according to SDS-PAGE
analysis. C-TRAF2 that was used in the NMR binding experiment was
concentrated to 41 mg/ml in 20 mM Tris, 200 mM NaCl, 10% glycerol,
20 mM DTT and 250 mM imidazole, pH 7.5.
[0067] NMR Spectroscopy: All NMR spectra were collected on a 600
MHZ Varian Unity Plus spectrometer. For the backbone assignments,
HNCACB and HN(CO)CACB experiments (Clore and Gronenborn, Methods
Enzymol. 239: 349-363, 1994; Muhandiram, et al., J. Magn. Reson.
B103: 208-216, 1994) were performed. To confirm the type of amino
acid assignment, C(CO)NH-TOCSY (Grzesiek, et al., J. Magnetic
Resonance, B101: 114-119, 1993) was used. Side chain resonances
were assigned from the following experiments: HBHA(CO)NH (Clore and
Gronenborn, Methods Enzymol. 239: 349-363, 1994), HC(CO)NH-TOCSY
(Grzesiek, et al., J. Magnetic Resonance B101: 114-119, 1993) for
the .sup.15N/.sup.13C sample in 90% H.sub.2O, 10% D.sub.2O,
HCCH-TOCSY for the .sup.15N/.sup.13C-labeled sample in D.sub.2O,
and .sup.15N-TOCSY-HSQC with the uniformly labeled .sup.15N
protein.
[0068] Initial backbone assignments were carried on with the
program ASSIGN (Lukin, et al., J. Biomolecular NMR 9: 151-166,
1997) with the HNCACB and HN(CO)CACB experiments. Stereospecific
assignments for .beta.-methylene protons and chi1 angles were
obtained from the HNHB (Archer, et al., J. Mag. Res. 95: 636-641,
1991), .sup.15N-TOCSY-HSQC and .sup.15N-NOESY-HSQC (Clore and
Gronenborn, Methods Enzymol. 239: 349-363, 1994) with a mixing time
of 40 ms. Stereospecific assignments of methyls in Leu residues
were obtained from the 3D .sup.13C-.sup.13C long range correlation
(Bax, J. Biom. NMR 4: 787-797, 1994), together with intra residual
NOE intensity.
[0069] Distance restraints were obtained from .sup.15N-Edited NOESY
at 50 ms and 100 ms, and .sup.13C-Edited NOESY at 80 ms
experiments. Due to the high overlap of methyl resonances, the
methyl-methyl NOE experiment at 90 ms mixing time (Zhwahlen, et
al., J. Am. Chem. Soc. 120: 7617-7625, 1998) was also performed for
the identification of NOEs in the methyl region. Slowly exchanging
amide protons were identified from a sample in 50% H.sub.2O, 50%
D.sub.2O by observing the intensity of the amide protons signals,
since N-TRADD could not be lyophilized to be redissolved in 100%
D.sub.2O. Half of an N-TRADD sample in H.sub.2O was diluted into
D.sub.2O, and a series of .sup.1H-.sup.15N HSQCs were taken to
monitor the decrease in intensity of the peaks. The other half was
diluted into a H.sub.2O buffer, and the .sup.1H-.sup.15N HSQC was
taken as reference. All peaks (50 amide protons) whose intensities
were not decreased by half (maximum decrease) after the first HSQC
(after 15 min of dilution in D.sub.2O) were used for the hydrogen
bond restraint. Phi angle restraints were obtained by measuring the
3J.sup.HN,H coupling constant from the HNHA experiment (Vuister and
Bax, J. Am. Chem. Soc. 115: 7772-7777, 1993).
[0070] Structures were calculated with a distance
geometry/simulated annealing protocol of XPLOR 3.851 (Brunger,
X-Plor 3.851: a system for X-ray Crystallography and NMR. Xplor
Version 3.851 New Haven, Conn.: Yale University Press, 1994),
adapted to incorporate secondary .sup.13C.alpha./.sup.13C.beta.
chemical shifts (Kuszewski, et al., J. Magn. Reson B106: 92-96,
1995) and a conformational data base potential for the non-bonded
contacts derived from high resolution x-ray structures (Kuszewski,
et al., Protein Science 5: 1067-1080, 1996), with 1873
proton/proton distance restraints, 100 hydrogen bond distance
restraints, 125 phi restraints, and 30 chi1 restraints. The NOE
distance restraints were categorized as strong (1.8-2.5 .ANG.),
medium (1.8-3.3 .ANG.) and weak (1.8-5.0 .ANG.).
[0071] C-TRAF2 and N-TRADD binding studies: The affinity of
wild-type N-TRADD and C-TRAF2 was measured by surface plasma
resonance using a BIAcore 2000 system (Pharmacia Biosensor AB).
C-TRAF2 (residues 262-501) was purified and coupled to a sensor
chip CM5 by amine coupling at pH=5.0 to get about 800, 1500 and
5500 response units on three different flow cells. A constant flow
(20 .mu.L/min) of purified N-TRADD protein in 10 mM Hepes, pH=7.4,
150 mM NaCl, 3.4 mM EDTA and 0.05% P20 surfactant at 8 different
concentrations from 650 .mu.M to 84 .mu.M was applied over the
protein coupled chip for 2 min to analyze the association. Binding
constants were obtained from the BIAevaluation software.
[0072] The CD40 peptide (SNTAAPVQETLHG-OH) was synthesized by using
fluorenylmethoxycarbonyl (Fmoc) solid-phase methods and purified by
reverse-phase HPLC.
[0073] Results and Discussion
[0074] Structure Determination: The structure of N-TRADD was
determined from 2302 NMR derived restraints obtained using
uniformly .sup.15N and .sup.15N/.sup.13C labeled protein, with
double and triple resonance NMR experiments. N-TRADD was soluble to
.about.1 mM, but high concentrations of dithiothreitol (20 mM) were
required to prevent aggregation of the protein. Under these
conditions the sample was stable for 6-8 weeks. The structural
statistics and root mean square deviations are shown below in Table
1. The atomic root mean square deviation about the mean coordinate
for residues 14-161 is 0.56 .ANG. for the backbone atoms, and 1.01
.ANG. for all atoms. For secondary structure elements only, the
rmsd is 0.46 .ANG. for backbone atoms and 0.92 .ANG. for all atoms.
The N-terminal residues 1-10 and C-terminal residues 162-169 are
disordered. The secondary structure for residues W11, V12 and G13
is not defined, due to lack of assignments for W11 (N, NH,
C.alpha., H.alpha.) and V12 (NH, N). The NH groups of these
residues were not observed, possibly due to conformational exchange
on the NMR time scale or to higher amide proton chemical exchange
rates. S14 is the first residue in .beta.-1, even though the N and
the NH assignments are missing. The evidence that S14 is part of
the beta strand is the presence of the characteristic interstrand
S14 (H.alpha.)-R76 (H.alpha.) NOE. The chemical shifts values for
C.alpha., C.beta. and H.alpha. are also indicative of a beta strand
structure for S14. This was also observed for R76, where only
C.alpha./H.alpha. and C.beta./H.beta. assignments were made.
[0075] Structure Description: The structure of N-TRADD consists of
5 alpha helices and four beta strands arranged in a novel fold.
N-TRADD has no sequence homology to any known protein based on a
BLAST search (Altschul, et al., Nucl. Acids. Res. 25: 3389-3402,
1997). Additionally, a structure similarity search was conducted
with DALI (Holm and Sander, Proteins: Structure Function and
Genetics 33: 88-96, 1998), and the structure of N-TRADD shows no
similarity to any of the distinct fold classes in the database.
[0076] N-TRADD is .about.40 .ANG. in length and .about.30 .ANG. in
width, where the four beta strands form an antiparallel beta sheet
composed of residues S14-E20, Q60-R66, L71-R76 and Q115-R119,
respectively, and the five alpha helices correspond to residues
L28-Y32, P35-G53, R80-L107, E132-A141 and E150-N161. The trace of
these structural elements is described as follows. The first beta
strand and a loop lead to helix 1, which is followed by helix 2 and
a three residue turn. .beta.-2, characterized by a beta bulge at
L62 and K63 and a beta hairpin (residues 67-70), is followed by
.beta.-3 and helix 3. Helix 3 is the longest helix, spanning nearly
the length of the protein and contains a slight curvature. The last
beta strand follows helix 3 and leads into a beta turn, a three
residue helical turn and helix 4. Finally, a stretch of eight
unstructured residues precedes the final helix. Helices 2 and 3
pack against the beta sheet, where most interactions are
hydrophobic in nature. Helix 4 makes contact with the N-terminus of
helix 2 and the N-terminus of N-TRADD, while helix 5 packs against
helix 1 and .beta.-2.
[0077] The hydrophobic core of the protein is formed primarily by
residues derived from all four beta strands (L17, V19, I64, V73,
L75, L116, L118) and residues in helix 1 (L28, Y32), helix 2 (V41,
L45, L49), and helix 3 (F87, Y90). Most hydrophobic residues are
buried in N-TRADD, except for a few residues in helix 3. V39 and
Y42 of helix 2 point away from the main core of the protein, making
hydrophobic contacts with residues in helix 5 (L152, L155 and
L159). Also, residues L62 and K63 in the beta bulge of .beta.-2 are
well positioned to make contact with helix 5. Interestingly, the
only tryptophan in N-TRADD, W11, is at the edge of the protein,
making long range hydrophobic contacts with L136, L139 and L140 in
helix 4.
[0078] NMR and BIAcore binding studies of N-TRADD with C-TRAF2: The
affinity of the interaction between N-TRADD and C-TRAF2 was
determined by BIAcore and the K.sub.d was found to be 6 .mu.M. The
.sup.1H-.sup.15N HSQC of N-TRADD was used as a tool to map the
binding interface with C-TRAF2, since the chemical shifts of
contact residues will be perturbed upon complex formation. In the
case of N-TRADD, the .sup.1H-.sup.15N HSQC peaks broadened with the
addition of C-TRAF2 due to formation of the large molecular size
complex. Differential line broadening was observed, which is
consistent with the expectation that the residues at the binding
interface would exhibit a larger resonance broadening when compared
to other residues in the protein. Equilibrium sedimentation
analysis (Park, et al., Nature 398: 533-538, 1999) has shown that
C-TRAF2 (310-501) is a trimer in solution, and both crystal
structures of C-TRAF2 (Park, et al., Nature 398: 533-538, 1999;
McWhirter, Proc. Natl. Acad. Sci. USA 96: 8408-8413, 1999) have
revealed a trimeric structure. The C-TRAF2 (residues 262-501) used
in the instant HSQC titration experiment (at 120 .mu.M) is also a
trimer, as determined by size exclusion chromatography. A surface
representation of residues in N-TRADD that exhibit broadened
.sup.1H-.sup.15N HSQC peaks by a factor of three or more relative
to the rest of the protein upon addition of C-TRAF2 indicate that
most of these residues are located on or nearby the beta sheet.
Many of the NH resonances belong to residues which face the
interior of the protein, and comprise part of the hydrophobic core.
The perturbed amino acids whose side chains face the surface of the
protein correspond to residues Y16 and F18 in .beta.-1, Q70, I72
and Q74 in .beta.-3, K63 and H65 in .beta.-2 and D68 in the hairpin
turn. Additionally, the NH of residues D149, E150 and L152 from
helix 5 are also significantly perturbed, as well as residues G121,
A122, R124 and L125 in the turn region between .beta.-4 and helix
4. The observed line broadening of these NMR resonances in the
.sup.1H-.sup.15N HSQC are most likely from residues in the binding
interface between N-TRADD and C-TRAF2. Additional residues with
observed chemical shift differences in their NH side chains include
L17, V58, L59, 164, V73, L75, C78, and L118. The side chains for
these residues are not facing the C-TRAF2 binding site of N-TRADD,
but exhibit significant perturburance when C-TRAF2 is nearby.
[0079] N-TRADD Mutagenesis and interactions between C-TRAF2 and
N-TRADD: In order to further characterize the amino acids in
N-TRADD that are involved in C-TRAF2 recognition, site directed
mutants of N-TRADD were prepared and evaluated for their ability to
bind C-TRAF2. Based on the NMR structure of N-TRADD and the
.sup.1H-.sup.15N HSQC analysis of the N-TRADD/C-TRAF2 interaction
described above, residues likely to be involved in the binding
interface were selected for mutagenesis studies. Five residues
(Y16, F18, H65, I72 and D149) were chosen which are located in
.beta.-1, .beta.-2, .beta.-3 and before helix 5. These residues
cluster on one side of the protein and therefore are most likely
involved in direct interactions with C-TRAF2 during complex
formation. In addition, mutations of these residues will be less
likely to affect the overall structure of N-TRADD. Indeed, this was
evident when the .sup.1H-.sup.15N HSQC spectrum of the mutant Y16A
was acquired and compared to the wild-type data. The
.sup.1H-.sup.15N HSQC for the mutant protein looks similar to the
wild-type protein, with chemical shift differences only for
residues near the mutation site (data not shown).
[0080] The affinity of N-TRADD mutants to C-TRAF2 was measured by
BIAcore. All mutant proteins showed a significant decrease in
binding affinity to C-TRAF2 when compared to wild-type N-TRADD. The
I72A and D149A mutant proteins show a modest effect, with a
.about.2-3 fold reduction in binding (FIG. 3), whereas F18A and
H65A mutant proteins show a large .about.7-10 fold decrease in
C-TRAF2 binding. The Y16A mutant protein shows the highest
reduction (.about.16 fold) in C-TRAF2 affinity. The mutagenesis
results in conjunction with the .sup.1H-.sup.15N HSQC perturbation
data suggests that N-TRADD residues in the beta sheet are essential
for C-TRAF2 binding. Residues Y16, F18, H65 in .beta.-1 and
.beta.-2 appear to be important in the interaction of N-TRADD with
C-TRAF2, leading to speculation that predominantly hydrophobic and
to some extent hydrophilic interactions may play a role in the
N-TRADD/C-TRAF2 complex formation.
[0081] Inhibition of N-TRADD/C-TRAF2 by CD40-derived peptide:
Recent reports on a C-TRAF2 binding peptide derived from CD40
receptor (Pullen, et al., Biochemistry 37: 11836-11845, 1998; Sato,
et al., FEBS Lett 358: 113-118, 1995; Nakano, et al., J. Biol.
Chem. 271: 14661-14664, 1996) led the inventors to study the effect
of this peptide on the N-TRADD/C-TRAF2 interaction. CD40 belongs to
the TNF receptor family and has been shown to interact with several
TRAF family members by yeast two hybrid analysis and
co-precipitation assays (Pullen, et al., J. Biol. Chem. 274:
14246-14254, 1999; Pullen, et al., Biochemistry 37: 11836-11845,
1998; Cheng, et al., Science 267: 1494-1498, 1995). In particular,
full length TRAF2 has been shown to interact directly with the CD40
cytoplasmic domain (Pullen, et al., Biochemistry 37: 11836-11845,
1998). The binding site of CD40 for TRAF2 was defined by peptide
mapping where the shortest CD40 sequence that TRAF2 recognized was
a five amino acid peptide with the sequence PVQET. The crystal
structure of C-TRAF2 (311-501) with a peptide derived from CD40
with sequence YPIQET (designated CD40-p1) (McWhirter, et al., Proc.
Natl. Acad. Sci. USA 96: 8408-8413, 1999) (Published with Protein
Data Bank at Accession No. 1QSC, and expressly incorporated herein
by reference) shows that it binds each of the TRAF2 monomers in the
C-TRAF2 trimer complex. Comparison with the structure of the
peptide from TNFR-2 in complex with C-TRAF2 (Park, et al., Nature
398: 533-538, 1999), which has a different consensus sequence
(QVPFSKEEC), reveals similar affinities and conformations
(McWhirter, et al., Proc. Natl. Acad. Sci. USA 96: 8408-8413,
1999). However, despite similar backbone contacts, the two peptides
are slightly shifted in the binding site, where CD40 peptide makes
many more complementary contacts with C-TRAF2 than does the
TNFR-2-derived peptide.
[0082] In the instant studies, the interaction between a 13-mer
peptide derived from CD40 (a longer version of CD40-p1, with
sequence SNTAAPVQETLHG) with C-TRAF2 was characterized, as well as
its effect on the N-TRADD/C-TRAF2 interaction. BIAcore studies show
that the peptide binds to C-TRAF2 with an affinity of .about.1.0 mM
(data not shown). In addition the peptide was also able to compete
for the binding of N-TRADD to C-TRAF2, with an IC50 of .about.1
mM.
[0083] Inhibition by the CD40-derived peptide was also observed in
the .sup.1H-.sup.15N HSQC studies of N-TRADD/C-TRAF2 binding. At
stoichiometric concentrations of N-TRADD and C-TRAF2 (110 .mu.M
each), HSQC peaks for N-TRADD have broadened out due to binding to
C-TRAF2 as described above. Addition of the 13-mer CD40 peptide at
a concentration of 3 mM restored the .sup.1H-.sup.15N HSQC spectrum
of N-TRADD alone (data not shown). These results are consistent
with the observed inhibition of N-TRADD/C-TRAF2 binding with the
CD40 peptide observed in the BIAcore experiments. The inhibition of
N-TRADD/C-TRAF2 by the CD40-derived peptide suggests overlapping
C-TRAF2 binding sites for CD40 and N-TRADD. No interaction between
N-TRADD and the CD40-derived peptide was observed, since no changes
in the .sup.1H-.sup.15N HSQC of N-TRADD were observed with addition
of the peptide. The lack of interaction between CD40 peptide and
N-TRADD was also observed in a BIAcore experiment, where the final
peptide concentration was approximately 15 mM (data not shown).
[0084] In addition to this 13-mer peptide, the effect of the
CD40-p1 peptide (McWhirter et al, Proc. Natl. Acad. Sci. USA 96:
8408-8413, 1999) on the interaction between N-TRADD and C-TRAF2 was
also evaluated. An inhibition with an IC.sub.50 around 200 mM was
observed, correlating with the reported K.sub.d for the binding of
CD40-p1 with C-TRAF2 (McWhirter, et al., Proc. Natl. Acad. Sci. USA
96: 8408-8413, 1999). The instant results suggest that the 13-mer
peptide also binds in the same C-TRAF2 groove as the 6-mer peptide,
a site also shared by N-TRADD.
[0085] Interaction interface between N-TRADD and C-TRAF2: The
N-TRADD/C-TRAF2 binding experiments, in conjunction with the CD40
peptide binding and inhibition data, suggest that the binding sites
on C-TRAF2 for CD40 and N-TRADD may overlap. The crystal structure
of C-TRAF2 complexed with the CD40 peptide (McWhirter, et al.,
Proc. Natl. Acad. Sci. USA 96: 8408-8413, 1999) show that each
C-TRAF2 monomer can bind to one peptide. The peptide binding site
in C-TRAF2 is located on the bottom and side of the `mushroom cap`,
covering roughly 500 .ANG..sup.2 of the C-TRAF2 surface and is
composed of hydrophobic and hydrophilic residues. The specific
interactions between the CD40 peptide and C-TRAF2 involve
hydrophobic contacts and a network of hydrogen bonds. Residues in
C-TRAF2 that make contact with the CD40 peptide in the crystal
complex (McWhirter, et al., Proc. Natl. Acad. Sci. USA 96:
8408-8413, 1999) include R393, Y395, D399, G400, F410, F447, R448,
P449, D450, S453, S454, S455, I465, A466, S467, G468, and P470.
[0086] Based on previous studies (Arch, et al., Genes Dev 12:
2821-2830, 1998; Park, et al., Nature 398: 533-538, 1999;
McWhirter, et al., Proc. Natl. Acad. Sci. USA 96: 8408-8413, 1999),
C-TRAF2 can recognize at least two different sequence motifs, SXXE
in the case of TNFR2 and PXQXT for CD40. Although the two peptides
make similar backbone contacts, each peptide makes different
additional unique contacts with C-TRAF2, suggesting the presence of
distinct recognition sites. Neither consensus sequence is present
in N-TRADD, implying that there may be another set of residues on
C-TRAF2 required for N-TRADD binding. This N-TRADD binding site on
C-TRAF2 would overlap with the CD40 binding site.
[0087] Conclusions
[0088] Based on the information obtained from these studies, the
presumed C-TRAF2 binding site in N-TRADD is comprised of residues
in one face of N-TRADD that are perturbed in the .sup.1H-.sup.15N
HSQC spectra upon addition of C-TRAF2 (Y16, F18, K63, H65, D68,
Q70, I72, G121, A122, R124, L125, D149, E150, and L152). Other
residues which are significantly perturbed in the .sup.1H-.sup.15N
HSQC spectra upon addition of C-TRAF2 but which have side chains
facing away from the presumed C-TRAF2 binding site include L17,
V58, V59, I64, V73, Q74, L75, C78, and L118. The N-TRADD binding
site in C-TRAF2, on the other hand, is based on the crystal
structure of C-TRAF2 with the peptide from CD40 (McWhirter, et al.,
Proc. Natl. Acad. Sci. USA 96: 8408-8413, 1999), and is presumed to
comprise residues R393, Y395, D399, G400, F410, F447, R448, P449,
D450, S453, S454, S455, I465, A466, S467, G468 and P470. A
comparison of the surface properties for the proposed binding sites
for N-TRADD and C-TRAF2 indicate that the surface of the binding
site for both molecules is not highly charged, suggesting that
their binding is based on hydrophobic interactions and is not
driven by electrostatic interactions. Consistent with this premise
is the observation that the N-TRADD/C-TRAF2 interaction is not
sensitive to NaCl (up to 1 mM, data not shown). This is not
surprising, since mostly van der Waals contacts and hydrogen
bonding are observed for both the TNFR-2 and CD40-derived peptides
complexes with C-TRAF2 (Park, et al., Nature 398: 533-538, 1999;
McWhirter, et al., Proc. Natl. Acad. Sci. USA 96: 8408-8413, 1999).
The fact that Y16, F18 and H65 mutations in the beta sheet of
N-TRADD displayed a large effect on the binding of N-TRADD with
C-TRAF2 suggests that these residues are important in the N-TRADD
affinity to C-TRAF2, presumably contributing a significant amount
of hydrophobic interactions between the two proteins. Several
residues on the C-TRAF2 binding site could potentially complement
these N-TRADD interactions, such as F410, F447, I465, Y395 and
P449. Both N-TRADD and C-TRAF2 binding surfaces are rich in
aromatic residues, suggesting that aromatic interactions, such as
ring stacking, may also contribute to their binding. These studies
provide a basis for further studies on the details of the
interaction between TRADD and TRAF2.
1TABLE 1 Structural Statistics and rmsds for the 25 NMR derived
structures for N-TRADD.sup.(1) Structural Statistics <SA>
<SA>r R.m.s. deviation from experimental distance restraints
(.ANG.) (2) All (1883) 0.022 .+-. 0.0013 0.022 Intraresidual (540)
0.010 .+-. 0.002 0.010 Sequential (506) 0.019 .+-. 0.002 0.016
Short range (387) 0.026 .+-. 0.003 0.026 Long range (450) 0.029
.+-. 0.002 0.031 R.m.s. deviation from experimental torsional angle
restraints (degrees) (3) .phi.(123), X1(31), X2(5) 0.21 .+-. 0.04
0.20 R.m.s. deviations from experimental 13C shifts (ppm) (4)
13C.alpha. (130) 1.14 .+-. 0.039 1.12 13C.beta. (130) 0.91 .+-.
0.019 0.96 R.m.s. deviation from idealized covalent geometry Bonds
(.ANG.) 0.003 .+-. 0.00009 0.003 Angles (.degree.) 0.60 .+-. 0.007
0.60 Impropers (.degree.) 0.40 .+-. 0.013 0.41 Ramachandram plot:
(5) Most favorable region: 91.1 .+-. 0.8 89.3 Gfactor 0.09 .+-.
0.01 0.08 N. bad contacts 5.3 .+-. 1.6 6 Cartesian coordinate
r.m.s. deviation (.ANG.) (6) Secondary structure Residues(14-161)
Backbone 0.46 0.56 Heavy atoms 0.94 1.01 Notes to Table 1 1.
<SA> is the ensemble of 25 NMR-derived structures, <SA>
r is the mean atomic structure obtained # by averaging the
individual SA structures (residues 14-161) followed by restrained
minimization. The # X-PLOR repel function was used to simulate the
van der Waals interactions with a force constant of 4.0 # kcal
mol-1.ANG.-4, with the atomic radii set to 0.8 times their CHARMM
PARAM19/20 parameters (REF). 2. The distance restraints were used
with a square-well potential (Fnoe = 30 kcal mol -1.ANG.-4). #
Medium-range NOEs are observed between protons separated by more
than one and less than five residues in sequence. # Long-range NOEs
are observed between protons separated by five or more residues. No
distance restraint was violated # by more than 0.30.ANG. in any of
the final structures. Hydrogen bonds were included as distance
restraints and # given the bounds of 1.8-2.3.ANG. (H--O) and
2.8-3.3 .ANG. (N--O). 3. The torsional restraints were applied with
a force constant of 200 kcal mol-1 rad-2, and no torsional
restraint # was violated by more than 5.degree. in any of the
structures. 4. The carbon chemical shift restraints were applied
with a force constant of 0.5 kcal mol-1 ppm -2. A conformational #
database potential based on the populations of various combinations
of torsion angles observed in a database of 70 # high-resolution
(1.75.ANG. or better) X-ray structures was used, with a force
constant of 1.0 (Kuszewski, et al, 1996) 5. The program PROCHECK
(Lakoswski, et al, 1993) was used to assess the quality of the
structures. 6. The precision of the atomic coordinates is defined
as the average rms difference between the 25 final calculated #
structures and the mean coordinates. The backbone atoms comprise of
N, C.alpha., C and O atoms.
[0089] All publications mentioned herein above, whether to issued
patents, pending applications, published articles, protein
structure deposits, or otherwise, are hereby incorporated by
reference in their entirety. While the foregoing invention has been
described in some detail for purposes of clarity and understanding,
it will be appreciated by one skilled in the art from a reading of
the disclosure that various changes in form and detail can be made
without departing from the true scope of the invention in the
appended claims.
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