helix that ended with residue T348. This helix
interacted along most of its length (R325 to
L344) with actin subdomain 1.
The structures of ABS1 and ABS2 were un-
equivocally overlaid onto the pointed end of the
actin filament (20), producing a model of the
pointed end that was then tested by mutagen-
esis (14). First, the structures showed that Tmod
could only bind at the pointed end, because
both ABS1 and ABS2 covered surfaces that are
buried in the filament. Second, ABS1 and ABS2
must bind to two different protomers at the
pointed end, because their binding surfaces par-
tially overlapped (fig. S3A). Third, the binding
surfaces of ABS1 and ABS2 also overlapped
when they were superimposed onto the second
and first protomers of the filament, respectively
(fig. S3B). This arrangement also placed the two
actin-binding sites asymmetrically on one side
of the filament, increasing the distance that
must be covered by the intervening sequence
(residues 100 to 170) that contains the second
TM-binding site. In contrast, no overlap was
observed when ABS1 and ABS2 were superim-
posed onto the first and second protomers of
the filament, respectively (Fig. 2A). This arrange-
ment naturally positioned ABS2 at the interface
between the first three subunits of the filament,
where Tmod residues in contact with actin were
highly conserved (fig. S4A). This model, and
P58-K99 R325-L344 Y170-N179 N201-R206 V232-R235 N258-F263 K286-S291 K314-H318
1 15 25 38 50 77 101109 126 144 160 359 349
S291-K297 R235 F263
194 223 251 279 309
Y87G W96G
F62D L67D R235E Q292E P293E
130°
P174G E176G E260G
Residues interacting with actin protomers 1, 2 and 3, respectively
Residues mutated in previous studies
F62
L67
T59-P61 L71
P174
E176
Y87
W96
K341-R343
R235
F263
Q292, P293
L313, K314
T59-P61
F62
Residues mutated here
A
337
322QGPR325 -> 322AAAA325, 322EEEE325
322QGPR325
Short Tmod isoform expressed in erythrocytes (residues M103–V359)
T320-R327
Actin-1
Actin-2
Actin-3
Actin-1
Actin-2
Actin-3
B C D
0
200 600 800
0.4
0.2
0.8
1.0
1.2
R
exp
/
Rc o
ntr
o
l
Tmod [nM]
KD = 107.8 nM
KD = 27.8 nM
0.6
1200 400 1000
Tmod100-359
TmodF62D, L67D
ABS1 mutants
Tmod Y87G, W96G
Tmod + TM
Controls
without TM
1.5 µM G-actin (6% pyrene-labeled), 1.5 µM phalloidin-stabilized F-actin, 25 nM CP, 1 µM TM
Tmod1-101
TmodP174G, E176G, E260G
TmodAAAA, TmodEEEE
322-325
ABS1
mutants
ABS2
mutants
200 nM Tmod
0
0.4
0.2
0.8
1.0
1.2
R
exp
/
Rcon
tr
o
l
0.6
R235E
Q292E
P293E
P174G
E176G
E260G
Y87G
W96G
WT actin 1-101 100-
F62D
L67D
Controls
AA
AA
EE
EE
0
100 200
0.4
0.2
0.8
1.0
1.2
Re x
p/
Rc
ont
ro
l
Tmod [nM]
0.6
400 300
AAAA
EEEE
V232
E260
Fig. 2. Testing the interactions of Tmod at the pointed end by mutagen-
esis. (A) Representation of the mutants studied here on a domain diagram of
Tmod and on the structures of ABS1 and ABS2 superimposed onto the first two
protomers (marine and blue) at the pointed end of the filament model (20) (see
also movie S3). Residues mutated here are colored by atom type (carbon,
yellow; oxygen, red; nitrogen, blue). Also shown are residues mutated in previous
studies (green). Note that ABS1 only interacts with the first protomer, whereas
ABS2 contacts the first three protomers of the filament. The third protomer is
colored gray, except for the area that contacts ABS2, which is colored purple.
(B) Normalized pointed-end elongation rates of filament seeds as a function of
Tmod concentration, with or without TM (conditions given on top). (C) Normalized
pointed-end elongation rates of filament seeds as a function of mutant Tmod
concentration, and in the presence of TM (the individual titrations are shown
in fig. S7). Each mutant is represented by a different color (keys are given on
the bottom). (D) Comparison of the normalized pointed-end elongation rates
at 200 nM Tmod with or without (gray bars) TM.
