at the pointed end (Fig. 3 and movie S4). ABS1
interacts only with the first protomer of the
filament (interface area: 1549 Å2) and ABS2 interacts mainly with the second protomer (interface
area: 1770 Å2), although it also contacts protomers 1 and 3 (estimated interface areas: 198 and
125 Å2, respectively). Secondary-structure prediction using several algorithms further suggests that
the two TM-binding sites have a related fold consisting of three helices (14). The model of the
pointed end proposed here is supported by analysis of several Tmod mutants and by steric constraints imposed by the structure of the filament
(20) and the blocked (22) and open (23) positions
of TM on the filament.
The structure of the pointed end–binding
VCD domain of Vibrio VopL was recently determined in complex with an actin trimer (25).
One of the subunits of the VCD dimer binds in
the same location as ABS2, i.e., at the interface
between the first three subunits of the filament
(fig. S8). VopL is a powerful nucleator, and this
activity is strongly enhanced by the presence
of actin monomer–binding WH2 domains N-terminal to VCD (26, 27). Similarly, the Tmod-related protein leiomodin is a powerful nucleator
(28), and this activity is enhanced by the presence
of a C-terminal tail that contains a WH2 domain.
Separately, ABS1 and ABS2 interact with mono-
meric actin with KDs of ~10 mM, independent
of the nucleotide state on actin, whereas full-
length Tmod binds to the pointed end with a
KD of 108 nM, and 28 nM in the presence of
TM. Thus, the increased affinity of Tmod at the
pointed end results from multiple, relatively
weak interactions, involving three actin proto-
mers and two TM molecules on each side of
the filament. We suggest that Tmod’s interac-
tions at the pointed end are better described
in the form of two TM-actin–binding modules.
Because of their relative independence and
weaker individual affinities, one module can
detach from the filament while the other re-
mains bound, which could explain pointed-end
subunit exchange in sarcomeres (13). Newly in-
corporated monomers likely consist of ATP-
actin, for which the two actin-binding sites of
Tmod appear to have an affinity similar to that
for ADP-actin, thought to be the predominant
species at the pointed end.
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Supported by NIH grant R01 GM073791. X-ray data collection at
beamline X6A of the National Synchrotron Light Source was
supported by NIH grant GM-0080 and U.S. Department of Energy
contract DE-AC02–98CH10886. We thank G. Rebowski for the
preparation of actin and TM for this study and W. Lehman and
M. Orzechowski for providing the TM models. The structures
have been deposited in the Protein Data Bank under codes 4PKG,
4PKH, and 4PKI.
Materials and Methods
Figs. S1 to S8
Movies S1 to S4
16 May 2014; accepted 24 June 2014