especially the location of the N termini of ABS1
and ABS2, was further consistent with the azimuthal sliding of TM on the surface of the
filament (fig. S5), which occurs upon Ca2+
binding to troponin and myosin binding to the filament (21). The two extreme positions of TM on
the filament, blocked and open, are represented
by electron microscopy (EM) structures of actin-TM (22) and actin-TM-myosin (23). The full
extent of TM’s sliding is estimated to be ~35°
(21), which greatly restricts the available surface
for Tmod binding at the pointed end and strongly supports the model proposed here.
Key interactions of Tmod with the first three
protomers of the filament (Fig. 2A and fig. S4)
were tested by mutagenesis and pointed end–
capping assays. In these assays, the barbed
end must be tightly capped, because faster dynamics at this end can mask subunit exchange
at the pointed end. Traditionally, gelsolin has
been used to cap the barbed end in Tmod studies
(7, 10, 12, 15–17). We found, however, that CP
and not gelsolin should be used in these assays
(fig. S6, A to D), because monomer sequestration
and filament severing by gelsolin interfered with
its barbed end capping activity (14).
The optimal concentration of TM for our experiments was 1 mM (14) (fig. S6, E to H). Thus,
the concentration dependence of Tmod capping
was determined in the absence or the presence
of 1 mM TM (Fig. 2B and fig. S7, A and B). TM
enhanced Tmod’s capping efficiency about fourfold, resulting in a KD of 28 nM compared to
108 nM in the absence of TM. These affinity
values could be directly compared, because the
same stock of filament seeds was used in the experiments. Generally, however, the affinity values
depend on the number of pointed ends, which
varies from experiment to experiment. Although
substantial, a ~4-fold increase in affinity was far
below the 1000-fold increase and picomolar affinity reported previously (7), which prompted
several repetitions of the experiments with identical results. A nanomolar rather than picomolar
affinity of Tmod at the pointed end seems more
consistent with the observation of pointed-end
monomer exchange in sarcomeres (13).
We analyzed eight Tmod mutants in elongation
assays (Fig. 2, figs. S4 and S7, and movie S3).
Erythrocytes express a short Tmod isoform (res-
idues 103 to 359) (24), which reinforces an idea
suggested by the structures; Tmod is better de-
scribed as consisting of two TM-actin–binding
modules. Thus, we expressed constructs 1 to 101
and 100 to 359 to test the importance of each
module. Other mutations were introduced within
full-length Tmod and targeted the a helix and
tail region of ABS1, and interactions of ABS2
with the first three protomers of the filament.
Although TM increased the capping efficiency
of the mutants, they all displayed reduced ac-
tivity compared to wild-type Tmod, supporting
the importance of these residues for Tmod-actin
interactions (Fig. 2, C and D, and fig. S7, C to J).
The most severe capping defects were observed
with the deletion or mutations of the C-terminal
TM-actin–binding module. Particularly, replac-
ing residues 322 to 325 at the beginning of the
C-terminal a helix with AAAA or EEEE had the
most pronounced effect, consistent with the key
location of this region at the interface between
the first three protomers of the filament. Muta-
tions reported to affect actin binding and capping
(9, 15–17) also tended to localize to the contact
surface with subunits of the filament (Fig. 2A,
fig. S4, and movie S3).
These results are consistent with a model in
which a single Tmod molecule caps actin filaments
Fig. 3. Model of the pointed end. (A) Domain diagram of Tmod (according
to Fig. 1A), showing a secondary-structure prediction of the N-terminal region
by several algorithms (14). The prediction suggests that the two TM-binding
sites, which are similar in size, also share a similar fold, consisting of a three-
helix bundle. (B) Model of the pointed end, with the structures of the com-
plexes of actin with ABS1 and ABS2 (magenta) superimposed onto the first
two protomers of the filament (marine and blue). ABS2 also contacts the
third protomer (purple-colored area). TM is shown in the stable “blocked”
position, which it assumes when bound alone to the filament (22). A tentative
model of the two TM-binding sites of Tmod (green), based on the results of
the secondary-structure prediction and energy minimization (14), is shown
for reference (see also movie S4).
