2 and 1 (Fig. 1, D and E, and movies S1 and S2).
An unusual feature of the structures was an interaction with the deoxyribonuclease I (DNase I)–
binding loop in actin. As a result, this loop, which
is disordered in most actin structures, was ordered in the Tmod complexes, albeit with different conformations in the two structures.
ABS1 extended beyond the a helix suggested
previously, with a total of 42 residues (P58 to
K99) mediating the interaction (Fig. 1, A and D).
ABS1 adopted a rather extended conformation
and contacted three out of the four actin sub-
domains. The a helix was longer than anticipated,
comprising residues 64 to 77. This helix played a
crucial role in the interaction, by forming a bridge
across the nucleotide-binding cleft on top of actin
subdomains 4 and 2. As a result, the nucleotide
cleft was slightly more closed in this structure
than in that of ABS2.
The interactions of ABS2 were also more ex-
tensive than anticipated and involved contacts
along the entire region Y170 to L344. Residues
Y170 to N179 formed a loop that interacted with
the DNase I–binding loop in actin. Several addi-
tional contacts were mediated by the LRR domain,
whose boundaries differed compared to the de-
scription of the structure of uncomplexed chicken
Tmod1 residues 179 to 344 (11). The LRR motif
is defined as a b-strand–loop–a-helix module (19).
The loop of the motif is called the “ascending
loop,” whereas the loop connecting one motif to
the next is called the “descending loop.” By this
criterion, Tmod contains not five but four-and-a-
half LRR motifs (Fig. 1A). The ascending loops
typically mediate protein-protein interactions (19),
and this principle was conserved in Tmod, where
the ascending loops interacted on the back side
(according to the classical view) of actin subdo-
mains 2 and 1 (Fig. 1 A and E). Most LRR do-
mains have N- and C-terminal caps, which shield
the hydrophobic core of the domain from solvent
exposure (19). In Tmod, these caps consisted of a
helices that ran diagonally to the first and last
LRR motifs: D182 to N193 and Q321 to N336. The
C-terminal cap continued as an uninterrupted a
P58-K99 R325-L344 Y170-N179 N201-R206 V232-R235 N258-F263 K286-S291 K314-H318
Leucine rich repeats within LRR domain
Fragments co-crystallized with actin N- and C-terminal helical caps of LRR domain
Helical segment within actin-binding site 1 TM-binding sites. Striped areas correspond to predicted helical segments
Regions observed in the structures Residues interacting with actin protomers 1 and 2, respectively
A
D
E
90°
90°
1152538506477 101109126 144160 359 349 194 223 251 279 309 337
DNase I-binding loop
0 50 100 150
Time (min)
0.0
3
-0.4
-0.6
-0.2
4 210
0
-2
-4
-6
D
if
fe
re
n
ti
al
p
ow
er
(µc
al
s
-
1)
H
ea
t
of
i
nj
ec
ti
on
(
kca
l
mo
le
-
1)
Molar ratio
N = 0.93
KD = 7.5 ± 0.47 µM
T = 10°C
ABS1 ATP-actin:GS1 B
C
0 50 100 150
Time (min)
0.0
1.5
-0.2
-0.1
2.0 1.0 0.5 0.0
-0.4
-0.8
-1.2
Di
ff
er
en
ti
al
po
we
r
(µc
al
s
-1
)
He
a
t
o
f
i
nj
ect
io
n
(kc
al
m
ol
e
-
1)
Molar ratio
N = 0.69
KD = 10.5 ± 0.83 µM
T = 10°C
0.0
ABS2 ATP-actin:GS1
2.5
DNase I-binding loop
1
24
3
3
1
2
4
321
Short Tmod isoform expressed in erythrocytes (residues M103–V359)
P58
K99
Y170
G349
ABS1 ABS2
Fig. 1. Structures of Tmod’s ABS1 and ABS2 bound to actin. (A) Domain
diagram of human Tmod1, showing the interactions of ABS1 and ABS2 with
actin. (B and C) Isothermal titration calorimetry (ITC) titrations of ABS1 ( 400 mM)
(B) and ABS2 (632 mM) (C) into 20 and 60 mM ATP-bound actin:GS1. For both
titrations, the best fit of the data corresponds to a one-site binding isotherm.
The reported errors correspond to the SD of the fits. (D and E) Two perpen-
dicular views of the structures of ABS1 (D) and ABS2 (E) bound to actin (blue).
GS1, fused N-terminally to the two Tmod domains, is omitted from this figure,
but shown in fig. S2. The side chains of Tmod residues that fall near actin are
shown (colored by atom type: carbon, yellow; oxygen, red; nitrogen, blue).
Circled numbers indicate actin subdomains 1 to 4. Note that subdomains 2 and
4 are exposed at the pointed end of the actin filament.
