Disabling TNF receptor signaling by induced
conformational perturbation of tryptophan-107
Ramachandran Murali*†‡§, Xin Cheng*, Alan Berezov*, Xiulian Du*, Arnie Scho ¨n¶, Ernesto Freire§¶, Xiaowei Xu*,
Youhai H. Chen*, and Mark I. Greene*†‡§
*Department of Pathology and Laboratory of Medicine and†Abramson Cancer Research Center, University of Pennsylvania, 36th Hamilton Walk,
Philadelphia, PA 19104; and¶Department of Biology, The Johns Hopkins University, Baltimore, MD 21218
Communicated by Peter C. Nowell, University of Pennsylvania School of Medicine, Philadelphia, PA, June 1, 2005 (received for review March 16, 2005)
We have disabled TNF receptor (TNFR) function by inducing allo-
steric modulation of tryptophan-107 (W107) in the receptor. The
allosteric effect operates by means of an allosteric cavity found a
short distance from a previously identified loop involved in ligand
binding. Occupying this cavity by small molecules leads to pertur-
bation of distal W107 and disables functions of the TNFR, a
molecule not known to undergo conformational change upon
binding TNF-?. TNF-?-induced NF-?B and p38 kinase activities and
clinical symptoms of collagen-induced arthritis in mice were all
diminished. Thus, disabling receptor function by induced confor-
mational changes of active binding surfaces represents an inno-
vative paradigm in structure-based drug design.
allosteric ? inhibitor ? drug design ? structure ? arthritis
various physical factors, including pH, solvents, ligand binding, and
oligomerization. Conformational changes can occur at a defined
local site or, as observed in multimeric proteins, at a distance from
the ligand-binding site (allosterism) (1–3). The basis and functional
importance of propagation of structural allosteric changes are
To our knowledge, altering a protein’s function by perturbing a
defined conformational state (i.e., not known to undergo confor-
mational change on activation) using rational structure-based de-
sign has not been achieved. Although surface cavities on nonen-
zymatic classes of proteins have been largely unexplored,
inactivation of enzymes has been accomplished by designing com-
petitive or substrate analog inhibitors that bind at active sites and
noncompetitive inhibitors identified generally by using high-
throughput screening. For example, extensive studies have been
reported on exosites (allosteric sites) of the thrombin–hirudin
complex (4, 5). Also, allosteric inhibitors of G protein-coupled
receptors (6) and muscarinic (7) and adenosine receptors (8) have
been found. Bodian et al. (9) have used the knowledge that
fusion-induced conformational change of influenza hemagglutinin
is required for virus entry and by a structure-based approach
the trimeric interface of the target protein.
Elements responsible for regulating conformational movements
in proteins are not fully understood. Little information exists
interface. The cavities’ relationship to inherent flexibility of sec-
ondary structural elements in proteins has not been explored. We
believe that cavities (internal and external) near critical secondary
elements may represent a significant feature related to protein
flexibility and function.
Based on these notions, we hypothesized that cavities and clefts
on the surface of proteins distal to regulatory sites, such as
have any discrete function might be used to modulate the function
of proteins?receptors by inducing conformational changes from
their native state. Induced allosterism would occur as a conse-
onformational flexibility is often essential for a protein to
function. Structural changes in proteins can be induced by
quence of lodging small molecules into the cavities. The mode of
inhibition is expected to share some features with that of allosteric
inhibitors, and hence the small molecules are referred to as
‘‘pseudoallosteric’’ inhibitors, the cavity is termed a ‘‘pseudoallos-
teric cavity,’’ and the method is described as ‘‘cavity-induced
allosteric modification’’ (‘‘CIAM’’). However, in our studies, the
pseudoallosteric sites are not known to be natural allosteric sites.
We have tried to define the basic principles by which predictable
conformational perturbation can be induced by selected com-
pounds. Our efforts have used well characterized receptor mole-
cules, which bind ligand by a structurally resolved mechanism but
are not known to have any allosteric site or allosterically mediated
function or undergo any conformational change on ligand binding.
The studies have focused on a class of previously unappreciated
surface cavities on the receptor ectodomain that are at defined
distances from the ligand-binding sites.
The TNF receptor (TNFR) is one of the central mediators of
inflammation (10). The three-dimensional structure of the TNFR1
complex has been determined with and without its ligand (11–14).
Active TNFR1 and TNF-? form a trimeric complex. The crystal
structure analysis of the TNFR complex with and without ligands
did not reveal any changes consistent with ligand-induced fit (13).
Hence, the structural role of the ligand was postulated to bring the
(G81 and G97) was also identified from the crystal structure
analysis (13), and it was predicted that this flexible hinge might
provide ligand-induced conformational changes. But, contrary to
that expectation, no significant conformational changes were ob-
served in the crystallographic complex (13). Here, we describe the
activity of a set of small molecules designed to bind to a discrete
surface cavity that disable ligand-induced TNFR functions as a
consequence of the defined conformational perturbation of tryp-
tophan-107 (W107) on the receptor. The conformational pertur-
biological activity in vitro and in vivo.
Materials and Cell Cultures. All chemicals were obtained from
Sigma. L929, THP1, and NE91 cells were all from the American
1640 (Invitrogen) containing 5% FBS. THP1 cells were cultured in
RPMI medium 1640 with the supplement of 50 mM Hepes?1 mM
Na pyruvate?50 ?M 2-mercaptoethanol?2.5 mg/ml glucose?50
?g/ml gentamicin?10% FBS. The cells were cultured in a 12-well
plate at a density of 6 ? 105cells per well with a supplement of 100
ng?ml phorbol 12-myristate 13-acetate. The cells were plated for
72 h to differentiate. NE91 cells were cultured in RPMI medium
1640 containing 10% FBS.
Abbreviations: CIA, collagen-induced arthritis; ITC, isothermal titration calorimetry; TNFR,
‡To whom correspondence may be addressed. E-mail: firstname.lastname@example.org or
§R.M., E.F., and M.I.G. have consulting agreements with Fulcrum Pharmaceuticals.
© 2005 by The National Academy of Sciences of the USA
August 2, 2005 ?
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no. 31 www.pnas.org?cgi?doi?10.1073?pnas.0504301102
WT and Mutant Human TNFR1 Cloning, Expression, and Purification.
The ectodomain of WT TNFR was obtained by PCR from pKP13
(15) (a gift from B. Beutler, UT Southwestern Medical Center,
Dallas) with 5? primer AAA AAA CAT ATG TAC CCC TCA
GGG GTT ATT GG and 3? primer CCG CTC GAG TCA ATG
and constructed into PET21 (Novagen) between NdeI and XhoT,
verified by sequencing. Mutant TNFR1 was obtained from site-
directed mutagenesis by using the QuikChange mutagenesis kit
(Stratagene). The plasmid was then transformed into Origami
(DE3) (Novagen). The WT and mutant TNFR1 were all expressed
in the inclusion bodies of the cells and were extracted and refolded
as described in ref. 16. The refolded protein solution was centri-
fuged at 14,000 ? g for 30 min at 4°C to remove the aggregation.
The supernatant was mixed with Talon metal affinity resin (QI-
Aexpressonst, Qiagen, Valencia, CA), rocked for 2 h at 4°C, and
with 50 mM NaH2PO4containing 300 mM NaCl and 150 mM
imidazole (pH 8.0).
Design of TNFR Binding Pseudoallosteric Ligand. A stepwise proce-
dure to identify pseudoallosteric sites and cavities used to induce
allosteric modification (a procedure termed CIAM) has been
developed (see Supporting Text, which is published as supporting
information on the PNAS web site).
Effect on TNF-?-Induced Cell Cytolysis.Themurinefibroblastcellline
L929 was maintained in RPMI medium 1640 supplemented with
5% FBS. L929 cells were seeded onto 96-well plates at a density of
The cells were then preincubated with designed small molecules at
the well at a final concentration of 100 pg?ml, and actinomycin D
optical density was then measured at 570 nm.
Effect on Regulation of Downstream Molecules of TNF-? Signaling.To
evaluate the effect of the designed small molecules on the regula-
tion of downstream molecules of TNF-? signaling, L929 cells (1 ?
106per well) were cultured in six-well plates for 12 h, treated with
at 20 ng?ml for the indicated periods. Cells were then washed with
separated by 12% SDS?PAGE, electroblotted onto nitrocellulose
membrane (Osmonics, Westborough, MA), and probed with anti-
phospho-I?B?, anti- I?B?, anti-phospho-p38, anti-p38, and anti-
?-actin antibodies (Cell Signaling Technology, Beverly, MA).
The membranes were then developed by using the enhanced
chemiluminescence (ECL) system (Amersham Pharmacia Bio-
sciences). For THP1 cells, differentiated THP1 cells (as described
under the heading ‘‘Materials and Cell Cultures’’) at day 3 were
pretreated with or without small molecules for 2 h and then treated
with TNF-? at 20 ng?ml at the indicated periods. Cells were then
blotting. To verify whether the effect on TNF-? downstream
signaling is specific for TNF-? signaling, NE91 cells were cultured
in a six-well plate at a density of 1 ? 106per well for 12 h. After 2 h
of treatment with or without designed small molecules, the cells
were stimulated with EGF at 50 ng?ml for the indicated time and
Fluorescence Quenching Studies. Quenchingexperimentswithacryl-
of 1 M acrylamide by adding 2.5 ?l of acrylamide each time.
Recombinant TNFR1 were at a concentration of 5 ?M in 1%
DMSO. Tryptophan emission, monitored at 340 nm, was observed
by using 295-nm excitation. Intensity data after quencher additions
were averaged over a 10-s collection and were corrected for
background emission (paired control lacking of protein). Intensi-
the Stern–Volmer equation,
F0?F ? 1 ? KS,V??Q?,
where F0is the emission intensity of the protein in the absence of
quencher and KSVis the Stern–Volmer constant for quenching,
given by the slope when data are plotted as F0?F versus [Q].
For synthesized small molecules, F002 at 20 ?M were prein-
cubated with 5 ?M TNFR1 in 1% DMSO for 30min and titrated
with 1 M acrylamide the same way as TNFR1 alone. The F0?F
was analyzed by using the Stern–Volmer equation, and two
slopes from TNFR and from TNFR with F002 were compared.
Kinetic Binding Studies by Surface Plasmon Resonance.Recombinant
TNFR WT and mutant were immobilized to the CM5 sensot chip
cavity is shown. TNFR1 is shown in a ribbon representation, and the allosteric
cavity is shown as a dot surface model rendered with INSIGHTII. The small
molecule, F002, is shown in a Corey–Pauling–Koltun (CPK) model to highlight
the volume of the pseudoallosteric cavity. The functionally critical residue
the target of conformational perturbation by F002.
Molecular model of the TNFR–F002 complex. A molecular model of
Murali et al.
August 2, 2005 ?
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of TNF-? to TNFR1 was estimated by using the Biacore 3000
instrument at 25°C. The apparent rate constants (konand koff) and
the equilibrium dissociation constant (Kd) for TNF?TNFR binding
by using BIAEVALUATION 3.0 software (Biacore).
Isothermal Titration Calorimetry (ITC). The binding thermodynamics
of inhibitors to the TNFR was measured by ITC using a high-
precision VP-ITC titration calorimetric system (Microcal LLC,
Northampton, MA). The calorimetric cell containing WT or mu-
tant TNFR at a concentration of ?1–6 ?M dissolved in 5 mM Tris
(pH 8.0) with 2% DMSO was titrated with the inhibitors dissolved
in the same buffer. The concentration of inhibitor was 50–120 ?M,
depending on the solubility in buffer. Injection volumes were 10 ?l.
All solutions were properly degassed to avoid any formation of
bubbles in the calorimeter during stirring. The heat evolved upon
each injection of inhibitor was obtained from the integral of the
calorimetric signal. The heat associated with the binding of the
inhibitor to TNFR was obtained by subtracting the heat of dilution
from the heat of reaction. The measurements were made at 25°C.
Data were analyzed and fitted by using the data analysis software
supplied by Microcal (ORIGIN 5.0).
Collagen-Induced Arthritis (CIA). Six- to 8-week-old male DBA?1
mice were purchased from The Jackson Laboratory and housed in
the University of Pennsylvania Animal Care Facilities. Animals
were maintained in accordance with guidelines of the Institutional
Animal Care and Use Committee (IACUC) of the University of
Pennsylvania. For CIA induction, mice were immunized by multi-
ple intradermal injections of 100 ?g of chicken type II collagen
antigen preparation i.p. on the 21st day. Mice were injected daily
with vehicle or F002 at different dosages (2 and 4 mg?kg of body
or toes; 2, erythema and mild swelling extending from the ankle to
the midfoot or ankle joint; 3, erythema and moderate swelling
extending from the ankle to the metatarsal joints; 4, erythema and
severe swelling encompassing the ankle, foot, and digits. The
maximum disease score per foot is 4, and the maximum disease
score per mouse is 16. For histological examination of the joint,
mice were killed at different time points, and their paws were
in hydrochloric acid, embedded in paraffin, sectioned, and stained
(55 ?M) binding to recombinant WT (A) and mutant (B) human TNFR1 at 25°C in 50 mM Tris?HCl buffer containing 2% DMSO is shown. F002 exhibited binding
affinity of 0.45 ?M and positive enthalpy change of 2.1 kcal?mol to WT human TNFR1; no binding affinity to mutant TNFR1 was exhibited.
Binding of F002 to TNFR1 by ITC. Direct binding of F002 to the soluble TNFR1 was measured by using ITC. Isothermal titration microcalorimetry of F002
loop. Stern–Volmer plots for quenching of the intrinsic tryptophan fluores-
cence of TNFR1 by acrylamide are shown. Aliquots of acrylamide at 1 M were
added to 5 ?M TNFR1 in PBS containing 1% DMSO. Parallel experiments were
carried out with 5 ?M TNFR1 plus 20 ?M F002 in PBS containing 1% DMSO.
Florescence emission at 340 nm was recorded at 25°C after excitation at 295
nm. The resultant concentration of quencher is shown up to 0.25 M, quench-
ing 77.4% of the total intrinsic fluorescence of TNFR1. The slope of TNFR1 is
14.4 ? 0.2 M?1; the slope of TNFR1 plus F002 is 11.6 ? 0.2 M?1.
Binding of F002 to TNFR induced conformational change in the WP9
www.pnas.org?cgi?doi?10.1073?pnas.0504301102Murali et al.
Identification of a Pseudoallosteric Cavities in TNFR. Earlier, in
TNFR1 we identified three critical contact sites (WP5, WP8, and
WP9) on the receptor needed for stable ligand complex formation
and found that WP9 (105–113) is the most critical loop for this
that if the configuration or disposition of the WP9 loop could be
perturbed, then the receptor’s structure would be such that it might
lead to a defective complex formation.
A site to perturb the WP9 loop was located by using an internally
developed a procedure called CIAM (cavity-induced allosteric
modification) (see Supporting Text). Using this algorithm, we iden-
tified a pseudoallosteric cavity distal to the WP9 loop. The algo-
cavities. To identify small molecule ligands that can be used to
induce conformational perturbation at WP9, standard virtual
screening procedures were used. Identified molecules were then
further selected and tested. Subsequently, active molecules were
further developed through synthetic modifications and evaluated
based on their ability to induce conformational changes, using
molecular simulation studies as a guide. These second-generation
molecules were used in the studies described below. The target
pseudoallosteric cavity in TNFR1 is shown in Fig. 1.
Binding Studies of Pseudoallosteric Inhibitors of TNFR. We evaluated
whether the second-generation compound F002 (Fulcrum Phar-
maceuticals, Las Vegas, NV) was able to bind to the isolated and
purified TNFR. First, we used surface plasmon resonance to
evaluate the kinetic binding of F002 to immobilized soluble TNFR.
Because of the inadequate sensitivity of this approach, the binding
results could not be interpreted unambiguously. ITC was used to
an affinity of 0.45 ?M. Binding was characterized by small positive
(unfavorable) enthalpy changes (?H ? 2.1 kcal?mol), indicating
that the interaction was entropically driven, as expected from a
binding reaction dominated by hydrophobic interactions. No bind-
ing could be observed for F001, a control compound with similar
mass and hydrophobicity to F002 but designed to be unable to
interact with the cavity.
Next, we determined that F002 actually binds to the pseudoal-
losteric cavity, using a genetic approach. To address this question,
we created mutants of the TNFR1 receptor in which the residues
(A) Inhibition of TNF-?-induced cytolysis. L929 cells were treated with human TNF-? and pseudoallosteric inhibitors at different concentrations. Absorbance
obtained without human TNF-? and with 1 unit?ml human TNF-? was used as reference for 100% survival and 0% survival, respectively. F002 exhibited the
inhibition dose-dependently; 10 ?g?ml F002 (or 27.2 ?M) shows ?50% inhibition of cytolysis caused by 1 unit of human TNF-?. (B) F002 attenuates
were pretreated with F002 at 20 ?g?ml for 2 h and stimulated by human TNF-? at 20 ng?ml for the indicated time. F002 reduced the phosphorylation level of
I?B and p38 at 5 min. Extracellular-regulated kinase 2 (Erk2) was analyzed as loading control. The relative phosphorylation intensity level of I?B? was 1.0, 2.2,
0.8, 0.3, 0.7, and 0.4, and the relative phosphorylation intensity level of P38 was 1, 4.5, 2.0, 1.3, 1.7, and 1.8, from left to right. The intensity was quantified by
NIH IMAGE software and normalized to the control Erk2 level. (C) Effect on the human promonocytic leukemia THP1. THP1 cells were pretreated with or without
F002 for 1 h and stimulated by human TNF-? at 100 ng?ml for 1 h. The results showed that F002 inhibited TNF-?-induced phosphorylation of I?B and p38 but
had no effect on LPS-induced activation. Lane 1, THP1 cells treated with vehicle; lane 2, LPS at 10 ng?ml; lane 3, TNF-? at 100 ng?ml; lane 4, F002 at 20 ?g?ml;
lane 5, LPS plus F002 at 20 ?g?ml; lane 6, TNF-? plus F002 at 20 ?g?ml. ?-Actin was analyzed as loading control. The relative phosphorylation intensity of I?B
normalized to the basal I?B levels is 1.0, 2.2, 1.2, 0.6, 1.4, and 0.5, and the phosphorylation intensity of p38 normalized to the basal p38 level is 1.0, 2.4, 2.2, 1.3,
2.3, and 0.9, from lane 1 to lane 6. The data were quantified by NIH IMAGE software. (D) F002 showed no effect on EGF-induced p38 activation in NE91 cells. NE91
cells were cultured in DMEM (10%) and pretreated with or without F002 at 20 ?g?ml for 2 h and stimulated by EGF at 50 ng?ml for 10 min. The cells were lysed
as loading control.
Murali et al.
August 2, 2005 ?
vol. 102 ?
no. 31 ?
we considered that charge introduction might limit the entropic
binding. We expected these changes to limit F002 access to the
cavity and to induce a change in disposition of the WP9 loop. The
structural integrity of the mutant receptor was verified by ligand
binding in surface plasmon resonance studies. TNF-? bound to the
WT TNFR1 (Kd? 3.79 ? 10?10M) and also to the recombinant
mutant TNFR1 (Kd? 0.45 ?M), suggesting that the mutations
affected the ligand-binding sites to some extent as expected. Using
ITC, we found that F002 no longer bound to the mutant receptor
(Fig. 2B) at all. These studies confirm that F002 bound to a single
and specific pseudoallosteric cavity as designed and that TNF-?
bound to the WT and the mutant receptor to a lesser extent.
Conformational Perturbation of the WP9 Loop in TNFR. Subsequently,
we evaluated whether the F002 binding involved conformational
changes in the WP9 loop, which had been shown to be critical for
TNF-? binding (17). Because there were no large detectable
conformational changes on ligand binding (13), we expected that
2 Å. Small conformational changes can be observed by x-ray
crystallography, but the technique requires high-resolution data.
Alternatively, fluorescence quenching can identify small modulat-
ing changes in proteins (18). Fortunately, only one tryptophan
residue exists in the TNFR1 ectodomain, located in the WP9 loop,
and we examined whether the tryptophan (W107) in WP9 under-
goes any change on F002 binding to the receptor. Fig. 3 shows the
results from fluorescence quenching induced by acrylamide. The
residue W107 in the WP9 loop fluoresces at ?340 nm. In this set
of experiments, the resultant concentration of quencher ranged up
to 0.25 M, quenching 77.4% of the total intrinsic fluorescence of
TNFR1. The Stern–Volmer constant for TNFR1 quenching by
acrylamide calculated from the slope of the plot is 14.4 ? 0.2 M?1,
compared with 11.6 ? 0.2 M?1for TNFR1 plus F002, indicating
that binding of FT002 to TNFR1 introduces conformational
changes in the TNFR1, which partly protects W107 from the
quencher. Some downward curvature in the Stern–Volmer plot
observed in the presence of F002 can be attributed to the existence
of two receptor populations, drug-bound and drug-free, with
different degrees of tryptophan exposure to the solvent (19, 20).
Thus, binding of F002 to the receptor changes the disposition of
tryptophan-107 from partially exposed to more completely buried.
We expected that this conformational change induced by F002
might lead to reduced affinity of ligand binding to the receptor and
might also create a defective receptor–ligand complex disabled for
In Vitro Effects of F002 on TNF-Mediated Signaling. Having estab-
lished that F002 bound to the pseudoallosteric cavity and induced
a conformational change, we next studied whether the F002-
function in vitro and in vivo. We examined changes in TNF-?-
mediated cytotoxicity in murine L929 cells. In this assay, F002
manner (Fig. 4A). Moreover, F002 binding inhibited TNF-?-
mediated I?B? and p38 phosphorylation in both murine L929 and
human monocyte cells, THP1 cells (Fig. 4 B and C), which are
thought to be key intermediaries of TNF-?-induced inflammation
in human cells.
As a critical control, we asked whether F002 could affect
EGF-stimulated cell signaling. EGF-mediated signals affect many
of the same pathways, and we sought to exclude the possibility that
the compound was acting through shared sites in the two signaling
pathways. We did not observe any F002-induced changes in EGF
signaling (Fig. 4D), supporting the notion that F002 specifically
alters the TNFR1 signaling pathways by binding to the receptor
ectodomain and inducing conformational changes. Alteration
rather than complete abrogation of the receptor’s function suggests
that overall structural integrity of the trimeric complex is intact.
Effect of F002 on CIA in Mice. Finally, we examined whether the
F002-induced conformational perturbation of W107 is important
for in vivo biological activity. Activity of F002 was studied in the
CIA system, which is a prototype animal model for human rheu-
matoid arthritis mediated by TNF-?. Six- to 8-week-old male
100 ?g of chicken type II collagen in 100 ?l of 0.1 M acetic acid
emulsified in an equal volume of complete Freund’s adjuvant and
were then challenged with the same antigen preparation i.p. on the
(2–4 mg?kg of body weight per day) from day 21 on. In this model,
and the severity can be determined by both physical examination
and joint histochemistry (21). Mice treated with F002 showed a
dose-dependent decrease in the clinical symptoms of arthritis
analysis of ankles of the animals revealed that F002-treated mice
have significantly reduced synovitis and mononuclear cell infiltra-
tion. Cartilage destruction was also prevented in the F002-treated
group (Fig. 5B).
Our previous efforts on targeted disabling of receptor ectodomains
by monoclonal antibodies or mimetics established the paradigm of
DBA?1 mice (n ? 6) were immunized with type II collagen to induce arthritis.
Twenty-eight days after immunization, mice were treated with vehicle and
in Experimental Protocol. Data are expressed as mean ? SEM.*, P ? 0.05;**,
P ? 0.01, by Student’s t test as compared with vehicle-treated control group.
(B) Joint histopathology at day 46. (a) The ankle joint from a normal DBA?1
mouse. (b) Severe inflammation and cartilage destruction occurred in a vehi-
cle-treated CIA mouse. Arrow indicates severe synovitis and erosion of carti-
lage. (c) Animals treated with 100 ?g?ml F002 show absence of synovitis in
ankle joints. (d) High-power view of the inflamed synovium in b.
F002 suppresses CIA in DBA?1 mice. (A) Inhibition of CIA by F002.
www.pnas.org?cgi?doi?10.1073?pnas.0504301102Murali et al.
selectively inhibiting receptor function by creating misaligned re- Download full-text
perturbation using small molecules that lodge in surface cavities
that reside distal to protein–protein interaction sites is an alternate
way to modify receptor function.
Mutational, crystallographic, and computer simulation studies
have suggested that a protein’s activity can be affected by changes
at the distal sites (4, 26, 27). In support of this hypothesis, myoglo-
bin, a multimeric protein, undergoes structural modification and
prosthetic group (28–31). Recently, tyrosine kinases and coagula-
tion-relevant serine protease factors have been found to have exo
sites (32, 33) similar to pseudoallosteric cavities defined in this
study. Finally, White and colleagues (34) have found that it is
possible to develop protein-interface binding molecules when the
proteins are known to undergo conformational change on oli-
gomerization. We have now demonstrated that in the absence of
ligand-induced conformational changes, alteration of flexible re-
gions by means of distal surface cavities is possible not only in
protein enzymes, but also in oligomeric complex receptors such
The relationship between protein function and the extent of
of whether inherent conformational plasticity can be exploited to
alter protein function is untested. Several crystallographic analyses
of protein complexes (35) reveal ligand-induced conformational
changes in the range of 2 Å or less, which are often unappreciated
and colleagues (36, 37) have recently suggested that small (?2 Å)
induced conformational changes in the aspartate receptor complex
may cause profound effects on receptor function. These types of
notions suggest that a small conformational change might be
sufficient to alter protein function. Our studies confirm these
observations. Small changes in the conformation of W107 are
sufficient to disable TNFR1 functions. Conformational changes
clearly can be induced either by point mutations or by small
molecules that are lodged near the WP9 loop. This study highlights
the role of inherent flexibility at the WP9 loop and the critical
nature of its disposition for TNFR function and suggests that small
molecules can be used effectively to perturb conformational ele-
ments critical for protein function and thereby disable function.
changes, and this evident from small positive (unfavorable) en-
thalpy changes (?H ? 2.1 kcal?mol), indicating that the interaction
was entropically driven, as expected from a binding reaction
dominated by hydrophobic interactions. Thus, binding of F002 to
the receptor cavity might involve displacing water molecules. Ear-
lier studies with the glucose binding yeast hexokinase implied that
entropic binding correlated with conformational changes in the
active site (38).
The F002 binding cavity is located away from the ligand inter-
but F002 and the subsequent movement of the WP9 loop does
affect ligand affinity for the receptor and limits effective ligand-
mediated signaling. Our view is that the F002-bound receptor is in
essence a defective complex that is less able to transduce signals in
an efficient manner. We suggest that F002 is not a competitive
inhibitor. It preferentially binds to the receptor before ligand
binding and hinders the formation of a stable complex. F002 might
also diffuse through the trimeric receptor complex and weaken
ligand interactions with the receptor. This latter scenario is indeed
possible, because the crystal structure of the complex reveals that
W107 is partially exposed to solvents (13).
Small conformational changes mediate receptor function in vivo.
It is not known whether small conformational changes in proteins
have any biological effect in mice. Point mutations can change
biological functions through small conformational rearrangements
at the location. Now, our studies suggest that small molecules can
also mimic point mutation in terms of conformational changes in
the receptor and alter protein function. Results from the CIA
model analyses reveal that small conformational perturbations can
that the receptor’s conformational integrity is critical for biological
function. These studies show that disabling receptor function by
conformational perturbation is effective for inhibiting arthritis in
In summary, these studies also indicate that certain but not all
receptor functions may be altered either positively or negatively by
discrete protein clefts and cavities located distant from the ligand-
binding sites. Further understanding of the relationship between
the clefts and cavities on the protein surface and inherent plasticity
of secondary structural elements can be exploited, and thus con-
an innovative component in structure-based rational drug design.
We thank Prof. Ray Ottenbrite and Dr. Ben Xiao (Fulcrum Pharma-
ceuticals) for providing F002 and Hongtao Zhang and Yuan Shen for
critical reading and helpful suggestions. This work was supported in part
by grants from the National Cancer Institute (to R.M. and M.I.G.) and
the National Institutes of Health (to M.I.G.) and by the Abramson
Family Cancer Research Institute (M.I.G.) and Fulcrum Pharmaceuti-
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