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INTRODUCTION |
TNF-
1 (cachetin) is a
pleiotropic cytokine with a variety of biological activities, including
cytotoxicity, immune cell proliferation, and mediation of inflammatory
responses (1). It exerts direct cytotoxic effects on a wide range of
human and murine tumor cell lines in vivo and in
vitro. TNF-
(lymphotoxin) is a lymphocyte-secreted cytokine
with a 32% identity in primary sequence to TNF-
(2). TNF-
exhibits pleiotropic activities similar to those of TNF-
(3, 4). In
contrast, the cellular origins, mechanism of induction, and mode of
secretion of TNF-
and TNF-
are different, and the two cytokines
produce different effects on several lymphoid, endothelial, and other
cellular targets (2, 5).
The active form of TNF-
and TNF-
appears to be a homotrimer. The
crystal structures of TNF-
and TNF-
revealed that each monomer
consists of two antiparallel
-pleated sheets with a jelly roll
topology and interacts with each other in a head-to-tail fashion to
form a homotrimeric structure (5-7). Diverse activities of TNF are
mediated by binding to each of the two receptors, TNF-R55 (55 kDa) and
TNF-R75 (75 kDa), on the cell surface. The Kd values
of TNF-R55 and TNF-R75 for the two TNFs are approximately 0.5 and 0.1 nM, respectively (4, 8). A number of TNF-
mutants were
reported that exhibited altered biological activities and receptor
binding affinities (9-14). These mutation studies showed that regions
corresponding to the lower part of each cleft between subunits are
important for the receptor binding as reviewed previously (14). Indeed,
in the crystal structure of TNF-
complexed with TNF-R55, these
regions interact with the receptor (15). The TNF and TNF-R55 complex
signals a large number of TNF activities, such as cytotoxicity,
manganese superoxide dismutase induction, fibroblast proliferation,
resistance to chlamidiae synthesis of prostaglandin E2, and
NF-
B induction (16-18), while the TNF and TNF-R75 complex is known
to transduce signals for the proliferation of primary thymocytes and T
cells (8, 19). In particular, signaling cascades emanating from
TNF-R55, which contains death domain, lead cells to apoptosis (6, 8).
TNF is also known to exhibit receptor-independent cytotoxic activity by
an ion channel formation at acidic pH. Under low pH that disfavors
receptor binding, the TNF trimer inserted in the cell membrane resulted
in the ion depletion of the cells, most likely through the hole in the
middle of the trimeric structure (20-22).
The clinical use of the potent antitumor activity of TNF-
has been
limited by the proinflammatory side effects including fever,
dose-limiting hypotension, hepatotoxicity, intravascular thrombosis,
and hemorrhage (23-26). Designing clinically applicable TNF-
mutants with low systemic toxicity has been an intense pharmacological interest (1, 23, 27). Human TNF-
, which binds to the murine TNF-R55
but not to the murine TNF-R75, exhibits retained antitumor activity and
reduced systemic toxicity in mice compared with murine TNF-
, which
binds to both murine TNF receptors (8, 19, 23, 28, 29). Based on these
results, many TNF-
mutants that selectively bind to TNF-R55 (L29S,
R32W, R32W/S86T, and E146K) have been designed. These mutants displayed
cytotoxic activities on tumor cell lines in vitro (23, 27,
30, 31), and exhibited lower systemic toxicity in vivo (29).
Reductions by up to 170-fold were observed for these mutants in
TNF-
's priming of human neutrophils for superoxide production and
antibody-dependent cell-mediated cytotoxicity, platelet-activating factor synthesis, and adhesion to endothelium (30).
In contrast, a human TNF-R75 selective mutant (D143F) did not show
lower proinflammatory activities (30), and another TNF-R75-selective
double mutant (D143N and A145R) did not exert cytotoxic responses in
human KYM-1 cells (27). The hypothesis that the preferential binding of
TNF-
to TNF-R55 is related to the low systemic toxicity, however,
was disputed in other studies. The R32W and S86T double TNF-
mutant,
which binds to TNF-R55 much more preferentially over TNF-R75 developed
serious systemic toxicity in a previous experiment in which healthy,
anesthetized baboons were infused with the TNF-
double mutant (31).
TNF-R55-deficient mice sensitized with D-galactosamine were
tolerant of endotoxic shock and insensitive to the TNF toxicities
(40-42). Thus, the role of TNF-R55 in the systemic toxicity needs to
be investigated further.
Recently, we have found that a human TNF-
mutant containing
substitutions of S52I and Y56F, plus a deletion of the N-terminal seven
residues, referred to as M3, exhibits increased binding affinities for
the two TNF receptors and increased in vitro cytotoxicity (32). When the L29S mutation was added, this TNF-
mutant, referred to as M3S hereafter, showed a lower and preferential binding affinity for TNF-R55 and substantially reduced systemic toxicity in mice while
inhibiting efficiently the growth of tumors transplanted on mice. M3S
displays increased thermal stability, resistance to the trypsin
proteolysis, and a longer half-life in vivo compared with
wtTNF-
. In addition, when combined with paclitaxel, M3S was found to
potentiate the antitumor efficacy of paclitaxel through the enhancement
of apoptosis (33). Interestingly, the design was recently reported of a
TNF-
mutant that exhibits low systemic toxicity but higher antitumor
activity than wtTNF-
and wtTNF-
(34). Although the structures of
TNF-
mutants are important for understanding their biological
activities, the three-dimensional structure of only one TNF-
mutant
(A84V) is reported. Here, we report the 1.8- and 2.15-Å crystal
structures of M3S in two different crystal packings and discuss the
mutational influences on the structure and biological activity of
TNF-
.
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MATERIALS AND METHODS |
Mutant Purification--
The Escherichia coli host
strain used in this study was BL21 (DE3) (F-ompT rB-mB-) whose
chromosome carries the T7 RNA polymerase gene under the control of
lacUV5 promoter. The gene encoding M3S was inserted downstream of T7
promoter to induce high level expression of the target protein. A
double plasmid system including a plasmid for a bacterial chaperone was
used to obtain M3S in a soluble form. Construction of these expression
vectors and purification schemes was described previously (35).
Cytotoxicity Measurement--
The cytotoxic activities of
wtTNF-
and M3S were measured on actinomycin D-treated murine L929
cells (ATCC CCL-929) according to the method described previously (36)
with slight modification. Briefly, L929 cells were seeded at 1 × 104 cells/well into a 96-well microtiter plate in
Dulbecco's modified Eagle's medium containing 2% fetal calf serum.
Eighteen hours later, medium containing 2 µg/ml actinomycin D was
added to the cell culture together with various concentrations of
wtTNF-
or M3S. The cells were incubated for an additional 18 h
at 37 °C. Cell viability was determined by measuring the cellular
metabolic activity with a 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl
tetrazolium bromide assay (37).
Systemic Toxicity Measurement--
Eight-week-old ICR mice (B & K) with an average body weight of 30 g were used for the systemic
toxicity measurement. Several doses of wtTNF-
or M3S were injected
intravenously. Lethality was checked 24 h after the single
administration, and the systemic toxicity was expressed as
LD50, a dose that kills 50% of the mice.
In Vivo Antitumor Activity--
A 0.1-ml aliquot of murine MH134
hepatoma cells (1 × 107 cells/ml) was transplanted
intradermally into the abdominal wall of 10-week-old female BALB/c
mice. In the case of murine Meth A fibrosarcoma, the same amount of
tumor cells were injected subcutaneously in the backs of 10-week-old
male BALB/c mice. When solid tumors of 3-7 mm in diameter formed,
wtTNF-
or M3S was administered intraperitoneally. Control mice
received saline in the same schedule for the experimental group. Each
group consisted of five mice.
Crystallization and Data Collection--
Two different initial
crystallization conditions were discovered using the screening method
of sparse matrix sampling (38). Refinement of the initial conditions
was carried out by using the hanging drop method, and two different
crystal forms of M3S were obtained. Crystals of type I (space group R3;
a = b = 66.72, c = 85.09 Å;
a monomer in the asymmetric unit) were obtained at 24 °C in the
mixture of 2 µl of protein solution (20 mg/ml, in 10 mM
HEPES buffer, pH 7.5) and equal volume of 2.8 M sodium
formate equilibrated with the same precipitant. Crystals of type II
(space group P212121;
a = 94.17, b = 94.56, c = 95.89 Å; two trimers in the asymmetric unit) were obtained in the
same way, but using different precipitant solutions containing 25%
polyethylene glycol 4000 and 0.1 M sodium citrate buffer,
pH 5.6. The crystallization conditions are totally different from those
of wild-type and A84V TNF-
mutant in which major precipitants were
magnesium sulfate and ammonium sulfate, respectively. The space groups
of the two crystals are also different from those of wild-type
(P41212) (5) and A84V mutant
(P3121) TNF-
. All x-ray diffraction data were collected
from single crystals with a MacScience DIP2020k imaging plate system
mounted on a MacScience M18XHF x-ray generator operated at 50 kV and 90 mA. Data for the type II crystal were collected from a frozen crystal
(110 K) soaked in the precipitant solution containing 28% polyethylene
glycol 400. All data were processed with the programs DENZO and
SCALEPACK.2 Table
I shows relevant data statistics.
Structure Determination and Refinement--
Structures of M3S
were determined using the 2.6-Å structure of wtTNF-
(PDB code 1tnf)
(5) by the molecular replacement protocols in the X-PLOR program
package (40). A monomeric model and trimeric model were used for the
type I and type II crystals, respectively. Peptide segments of residues
19-25, 29-36, 67-73, and 145-150 in each subunit were omitted
throughout the molecular replacement procedure, since they contain
errors as described in the coordinate description probably due to weak
electron densities in these regions. In both cases, strong peaks were
observed in the Patterson correlation refinements of the rotation
function peaks (41), which were at least 2 times higher than the next highest peaks buried in the noise level. These refined rotation function peaks yielded correct top translation function peaks as judged
by the low R-factors of 32.2% for the type I and 34.8% for
the type II crystal form (two trimers combined). With the omission of
the "problematic regions" mentioned above, a rigid body refinement
and an atomic position refinement against reflections at 10-3.0-Å
resolution were carried out for each of the translation solutions,
resulting in the R-factor of 26.5 and 27.0% for the type I
and type II crystal form, respectively. Even at this stage, strong
electron densities for the two omitted regions, residues 29-36 and
145-150, were observed. Multiple rounds of iterative manual refitting
of many main chains and side chains and incorporation of water
molecules were carried out according to the
2Fo-Fc and Fo-Fc electron density
maps while gradually increasing the resolution limits to maximums. The
current models have reasonable geometry as shown in Table I.
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RESULTS AND DISCUSSION |
Biological Properties--
M3S displays unusual biological
properties. The in vitro cytotoxicity of M3S is reduced to
5% of wtTNF-
, and the in vivo systemic toxicity of M3S,
which is indicated by LD50, is 10 times lower than that of
wtTNF-
in a lethality test using
mice.3 However, compared with
wtTNF-
, a substantial delay in the tumor growth was observed in mice
bearing transplanted Meth A fibrosarcoma or MH134 hepatoma solid tumor
after M3S was administered intraperitoneally every other day for 10 days.3 TNF exhibits biological activities mainly through
the interactions with the receptors; thereby the distinctive cytotoxic
properties of M3S should be directly related to altered interactions
with the receptors. M3S exhibits a lower and selective binding affinity for TNF-R55. The affinity for TNF-R55 and TNF-R75 is 11- and 71-fold lower, respectively, compared with wtTNF-
. M3 displayed increased binding affinities for both the receptors by 13- and 11-fold, respectively, and exhibited an increased cytotoxicity.3
Therefore, in M3S, the I29S mutation counteracts the increased receptor
binding affinities by the mutations of S52I and Y56F.
The deletion of up to eight N-terminal amino acids has been known to
increase the activity of TNF-
by a factor of 1.5-5, as measured by
an L929 cytotoxicity assay (9, 14, 42-44). Our independent assessment
of the L929 cytotoxic activity of a mutant lacking the N-terminal seven
amino acids showed that the in vitro cytotoxicity increased
by only about 20% compared with wtTNF-
. Therefore, the deletion
minimally affects the cytotoxicities of M3S and M3.
Large Local Conformational Change by the L29S
Substitution--
The three mutation sites are located at the bottom
of the bell-shaped structure of TNF-
as shown in Fig.
1. The structures of M3S reveal the first
close-up view of the consequence of L29S mutation in TNF-
, which has
been studied extensively due to the preferential binding affinity of
the mutant for TNF-R55 (1, 12, 45). While the loop containing residues
29-36, referred as segment A hereafter, is poorly defined in the
structure of wtTNF-
, strong electron density of this region was
observed for the structures of M3S in the two different crystal
packings (Fig. 2, A and
B). The mutation induces the formation of a hydrogen bond
between the Ser29 OH and the amide nitrogen of
Arg31, which is accompanied by many significant changes
(Fig. 2C). The peptide bonds between Asn30 and
Arg31 and between Ala33 and Asn34
rotate almost 180° compared with the wtTNF-
and A84V TNF-
mutant. As a result of the peptide bond rotations, the side chain of
Asn34 moves significantly to form favorable interactions
with neighboring residues. These are parts of the intricate
interactions of a hydrogen-bonded network involving Asn34,
Ala35, four residues from the adjacent subunit
(Arg82, Gln125, Glu127, and
Asp130), and a cluster of seven water molecules (Fig.
3). The O
1 atom of
Asn34 forms hydrogen bonds with the guanidino group of
Arg82 in the adjacent subunit and a tightly bound water
molecule (B-factor = 18.42), which is in a hydrogen
bond distance from the amide oxygen of Gln125 in the
adjacent subunit (Fig. 3). The favorable intra- and intermolecular interactions in M3S, which are not observed in the structure of wtTNF-
or A84V mutant, are consistent with the substantially lower
temperature factors of the atoms in segment A than those of wtTNF-
(Fig. 4). Since mutations in segment A
are known to affect the biological activities of TNF-
(1, 12, 14,
45), the large local conformational change induced by the L29S mutation as well as the amino acid difference itself should be responsible for
the altered binding affinities for the TNF receptors and the in
vivo activities of the mutant.

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Fig. 1.
Ribbon drawing of M3S trimer. Each
subunit is represented by yellow, red, and
magenta, respectively. The three mutated residues are in
green ball-and-stick models. Ser29 is located on
a loop. Ile52 is at the start of a loop, and
Phe56 is at the beginning of a -strand.
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Fig. 2.
Stereo diagrams of segment A (residues
29-36). A, the 1.8-Å
2Fo-Fc electron density map of the
type I crystal. B, the 2.15-Å
2Fo-Fc electron density map of the
type II crystal. The maps are contoured at 1.0 level. For clarity,
the side chains of Arg31 and Arg32 are omitted.
Two water molecules are shown at the same positions in the two
different crystal forms. C, segment A in the structures of
M3S (green) and of A84V TNF- (tan). For
clarity, the side chain of Arg31 is omitted. The
white dotted line indicates a hydrogen bond. Oxygen and
nitrogen atoms are in red and yellow,
respectively. The structure of wtTNF- was not used because of the
coordinate errors in this region. Backbone atoms of residues 8-157 in
both structures except for residues 71-72, 84-91, and 105-110 are
superimposed.
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Fig. 3.
A hydrogen-bonded network induced by the I29S
mutation. White dotted lines indicate hydrogen bonds. Water
molecules are in magenta. Oxygen and nitrogen atoms are in
red and yellow, respectively. Each subunit is
represented by green and pink, respectively. Only
the side chains involved in the interactions are shown for clarity.
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Fig. 4.
The temperature factors of the main chain
atoms in the structures of wtTNF- (broken line) and M3S
(solid line). Left and right arrows
indicate segment A and the loop containing Arg44,
respectively.
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Superposition of 1.8- and 2.15-Å structures (root mean square
deviation of 0.3 Å for backbone atoms) reveals virtually the same
conformations of segment A, indicating that the observed conformational
change is solely induced by the mutation (Fig. 2, B and
C).
Mutational Effect of S52I and Y56F Substitutions--
The
conformation of the loop containing mutations, S52I and Y56F,
introduced to both M3 and M3S is unambiguously determined. Since
Ser29 is far apart from these two residues, the
conformation of this region would not be affected by the L29S mutation
and should be the same in both M3 and M3S. The double mutations do not
cause a noticeable conformational change of this loop region. The
hydrophobic side chain of Ile52 is exposed to the solvent,
and Phe56 is buried completely in M3S as Ser52
and Tyr56 are in the structure of wtTNF-
(Fig.
5). Phe56 is involved in
hydrophobic interactions with Pro12, Val41,
Ile52, Leu126, Ile154, and
Ala156. Tyr56 is an important residue in the
interactions between TNF-
and its receptors (5), because several
mutants (Y56N, Y56A, and Y56S) displayed impaired or reduced receptor
binding and cytotoxic activities (13). The role of Tyr56 in
the receptor binding, however, has not been satisfactorily explained.
Tyr56 is not likely to interact with the receptors
directly, since the residue is buried completely in the hydrophobic
cavity. It was noted that Phe56 (or Tyr56 in
wtTNF-
) is surrounded by the loop containing residues 50-54, and
thus the residue appears important for propping the structure of the
loop (Fig. 5). The loop is likely to interact with the receptors
directly, since the S52I mutation in M3 increases receptor binding
affinity (32). The substitutions of Tyr56 with a small
amino acid, such as Y56A and Y56S, would not support the loop in the
proper position for the interaction with the receptor. A similar
observation was made in the structure of rat IgG where Phe27 contacts with the hypervariable loop (residues
31-35) important for interactions with an antigen (46). When
phenylalanine was replaced by serine, the affinity of the antibody was
reduced drastically because the tight packing of the hypervariable loop
was disrupted (47).

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Fig. 5.
The local environment of the loop containing
residues 50-56 in M3S. Thick white lines indicate the side
chains of Ile52 and Phe56. Green
dots are the solvent-accessible surface of Ile52, and
white dots are the 200% van der Waals radii of the atoms in
Phe56.
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Other Structural Features--
The current refined 1.8-Å
structure, with coordinate errors less than 0.3 Å, provides much more
detailed structural features than the wtTNF-
structure at 2.6 Å and
A84V TNF-
mutant structure at 3.0-Å resolution. While the structure
of wtTNF-
does not contain any water molecule, a total of 144 water
molecules are found in the 1.8-Å structure of M3S trimer, some of
which are involved in the intersubunit interactions. Nearly the same
disposition of water molecules is found (Fig. 2, A and
B) in the 2.15-Å structure except in the crystal packing
interfaces. The backbone atoms of the M3S structure superpose on those
of the wtTNF-
structure with the root mean square deviations of 0.66 Å. Relatively large deviations were observed in the loops containing
residues 67-73, 84-88, 105-110, 144-150, and segment A, which are
poorly defined regions in the wtTNF-
structure. While weak electron
densities for residues 67-73, 84-88, and 105-110 were also observed
in the structures of M3S in the two different crystal packings, strong electron densities for segment A and residues 144-150 were observed. The two loops are located near the trimer interface, and receptor binding studies suggested that the loop containing residues 144-150 is
also involved in the receptor binding (1, 13, 14, 23, 27, 45). Compared
with the structure of wtTNF-
, a hydrogen-bonded network is found
along this region, which is composed of five tightly bound water
molecules, His15, Ala146, Ser147,
and two residues belonging to the adjacent subunit (Asn92
and Leu93) (Fig. 6). These
extensive interactions should be responsible for the ordered structure
of the loop. In the wtTNF-
structure, Arg32 is involved
in a "putative" salt bridge with Glu146 and a hydrogen
bond with Ser147 (5, 13). In contrast, Arg32
interacts with the carbonyl oxygen of Phe144, and
Ser147 interacts directly with Asn92 of the
adjacent subunit in the M3S structure (Figs. 6 and
7). The peptide bond between
Phe144 and Ala145 is flipped, and the side
chain of Phe144 is buried more as indicated by the smaller
solvent-accessible surface of the residue (33 Å2) than
that of the residue in the wtTNF-
structure (66 Å2;
average value of three subunits) (Fig. 7). In this conformation, the
flipped peptide bond orients the carbonyl oxygen of Phe144
in the proper position to interact with Arg32 via a weakly
bound water molecule (B-factor = 32.75), and the side
chain of Phe144 interacts with Ala18,
Leu142, Pro20, and Val150, none of
which come in contact with Phe144 in the structure of
wtTNF-
except in one subunit. The one contact between residues
144-150 and segment A via the weakly bound water molecule would hardly
be responsible for the local conformational difference of residues
144-150 between wtTNF-
and M3S. Rather, the extensive interactions
are also present in the structure of wtTNF-
, and the conformation of
the residues 144-150 observed in the M3S structure is the correct
conformation.

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Fig. 6.
Interactions of His15 with
neighboring residues. White dotted lines indicate hydrogen
bonds. Oxygen and nitrogen atoms are in red and
yellow, respectively. Water molecules are in
magenta. Two adjacent subunits are represented by
green and pink, respectively.
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Fig. 7.
Interactions of Phe144 and
Arg32 with neighboring residues. The white
dotted line indicates a hydrogen bond. Oxygen and nitrogen atoms
are in red and yellow, respectively. A water
molecule is in magenta. The side chains except for those
involved in the interactions are omitted for clarity.
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Previously, the analyses of the cytotoxic activities of TNF-
mutants
containing a substitution of His15 (H15Q, H15N, and H15K)
suggested that the residue forms an important hydrogen bond critical
for the formation of a bioactive conformer (48). Consistently, the
imidazole ring nitrogen of His15 interacts with the
carbonyl oxygen of Leu93 in the adjacent subunit via a
water molecule (B-factor = 13.06) in the
hydrogen-bonded network (Fig. 6). The high resolution structure also
shows clearly that the His15 is a buried residue (solvent
accessibility of 2 Å2) with its imidazole ring involved in
a hydrogen bond with the hydroxyl group of Tyr59 and in an
aromatic ring stacking with Phe124 of the adjacent subunit.
These features indicate that His15 should be a structurally
important residue, and mutations of this residue could result in
biologically inactive TNF-
molecules.
Resistance to Proteolysis, Thermal Stability, and Longer
Half-life--
The limited proteolysis of wtTNF-
by trypsin is
known to cleave after Arg6 and Arg44, while
wtTNF-
remained intact following incubation with the SV-8 protease,
which recognizes negatively charged amino acids (49). As shown in Fig.
9, M3S is much more resistant to the proteolysis by trypsin compared
with wtTNF-
. Superposition of the structures of M3S and wtTNF-
showed that the local environment of the region containing
Arg44 is nearly identical. Leu43 participates
in the hydrophobic interaction with Leu37,
Val16, Val41, and Leu48 (Fig.
8). Asn46 is involved in two
hydrogen bonds with the carbonyl oxygen of Leu26 and
the amide nitrogen of Trp28 (Fig. 8). These
interactions account for lower main chain B-factors of
residues 43-46 in the structure of both wtTNF-
and M3S compared with other surface-exposed loops (Fig. 4). Thus, it was quite surprising that this rigid loop was reported to be susceptible to
trypsin while Arg31-Arg32-Ala33
(RRA sequence) on the highly disordered segment A in wtTNF-
was not.
However, it was noted that a short fragment, which might be generated
from the cleavage after Arg31 (or Arg32) and
Arg44, could not be detected because the digestion mixture
was dialyzed before SDS-PAGE, which identified only the large fragments
of residues 7-157 and residues 45-157 along with N-terminal
sequencing (49) (Fig. 9). Since the RRA
sequence is close to the hydrogen bonds and the hydrophobic
interactions involving the loop containing Arg44 (Fig. 8),
we assumed that the sequence is cleaved first by trypsin, and then the
cleavage could result in the disruption of these interactions. As a
result, the loop containing Arg44 could become flexible and
susceptible to trypsin. To confirm this sequential cleavage, we
analyzed the peptide fragments generated by trypsin digestion of
wtTNF-
using high performance liquid chromatography-mass
spectrometry.4 In the
analysis, the peptide fragments corresponding to residues 7-31
(calculated Mr = 2756.1; observed
Mr = 2756.5) and residues 33-44 (calculated
Mr = 1240.4, observed Mr = 1240.0) were indeed detected. However, the fragments corresponding to
residues 7-44 were not detected, supporting the idea that the cleavage
at Arg31 or Arg32 precedes the cleavage at
residue 44. The peptide fragments corresponding to residues 33-157
were not detected by either the mass spectrometric analysis or the gel
electrophoresis, indicating that the cleavage at residue 44 occurs
immediately after the cleavage at Arg31 or
Arg32. The identification of the major cleavage sites is
consistent with the resistance of M3S to trypsin digestion. Since
segment A containing the RRA sequence in M3S is rigid compared with
wtTNF-
(Fig. 4), the three arginine residues would not be easily
accessible to the cleavage by trypsin.

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Fig. 8.
Stereo diagram of the interactions of the
loop containing Arg44 and of the loop containing
Arg31 and Arg32. Arginine residues are in
magenta, and hydrophobic side chains are in
green. White dotted lines indicate hydrogen
bonds. Oxygen and nitrogen atoms are in red and
yellow, respectively. Only the side chains involved in the
interactions are shown for clarity.
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Fig. 9.
SDS-PAGE of 48 h time course trypsin
digestion of wtTNF- and M3S. The cleaved products are the same
bands observed in the early study (49) and correspond to the fragments
of residues 7-157 (upper band, residues 8-157 in the case
of M3S) and residues 45-157 (lower band). TNF and trypsin
were mixed (100:1, w/w) and incubated at 4 °C for 48 h. A
20-µl aliquot was sampled at each time point and mixed with SDS-PAGE
buffer containing 0.125 M Tris-HCl, pH 6.8, 2.15% SDS,
0.005% bromphenol blue, 20% glycerol, and 5% -mercaptoethanol.
The mixture was subjected to SDS-PAGE on 14% polyacrylamide gels.
Lane M indicates size markers. Lanes 1-9 indicate the incubation times in hours. Lane 1, 0;
lane 2, 0.5; lane 3, 1; lane 4, 3;
lane 5, 5; lane 6, 7; lane 7, 9;
lane 8, 24; lane 9, 48.
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M3S exhibits increased thermal stability and a longer in
vivo half-life. After incubation at 25 °C for 24 h, 2 times more activity was retained in M3S compared with wtTNF-
in L929
cytotoxicity assay.3 The in vivo half-life of
wtTNF-
, which follows a pseudo-zero-order reaction, was 29 min at a
dose of 500 µg injected into experimental rats. In comparison, the
half-life of M3S was increased to 54 min at a significantly lower dose
of 100 µg in the same animal model system. The I29S substitution
should confer the thermal stability of M3S, which not only reconstructs
segment A into a rigid segment but also increases the intersubunit
interactions. Since active trimeric TNF-
slowly converts into
inactive monomeric forms (50), the increased thermal stability and
resistance to proteolysis of M3S could be the reasons for its longer
in vivo half-life. It was confirmed that M3 does not show an
increased thermal stability.3
Modulation of Cytotoxicity--
The receptor-independent
cytotoxicity of TNF mediated by the ion channel-forming activity at low
pH (39, 51) requires structural changes. It has been demonstrated that
structural plasticity of the TNF-
trimer is a major determinant of
its channel-forming activity, and that Gly23,
Arg32, and Arg44 become highly accessible to
proteases Arg-C and V8 after membrane insertion (39). Interestingly,
Gly23, Trp28, Arg32, and
Arg44 are on or near segment A, implying that the flexible
structure of segment A in wtTNF-
may play a key role in the ion
channel formation. Thus, M3S is not likely to undergo structural
changes essential for the ion channel formation as easily as wtTNF-
due to the rigidity of segment A. It is possible that the increased stability of M3S could result in a lower receptor-independent cytotoxicity.
Taken together, the desirable low systemic toxicity of M3S is likely to
be the effect of the reduced binding affinities for the two TNF
receptors with the preferential binding affinity for TNF-R55, and
possibly of a reduced receptor-independent cytotoxicity in
vivo. The superior tumor-suppressing activity of M3S, despite its
lower cytotoxicity, is likely to be the result of the extended antitumoral activity due to its longer in vivo half-life.
The concept of attenuated but prolonged biological effects of TNF-
in vivo may be a key for designing therapeutically valuable
TNF molecules.
In summary, the comparison of the crystal structures of M3S and
wtTNF-
reveals that 1) the L29S mutation results in favorable intra-
and intersubunit interactions, which should be responsible for the
increased thermal stability, resistance to proteolytic cleavage, and
altered receptor binding affinities; 2) the I52S and Y56F mutations do
not induce a noticeable conformational change; 3) substituted
Ile52 is exposed to the bulk solvent and is likely to
interact with the TNF receptors favorably as it is supported by the
increased binding affinities of M3 for the receptors. The structural
analyses of M3S structures led to the identification of the major
proteolytic cleavage sites at
Arg31-Arg32-Ala33 by a mass
spectrometric method. The analyses also provided structural bases for
the previous conclusions that His15 and Tyr56
are important for the bioactive conformation and/or the receptor binding. It is also anticipated that the high resolution structure of
M3S will serve as a better model to explain the structure-function relationship of wtTNF-
and many other TNF-
mutants generated so
far and for the design of other TNF-
mutants with therapeutic potentials.