Structure of the light chain-binding domain
of myosin V
Mohammed Terrak*, Grzegorz Rebowski*, Renne C. Lu, Zenon Grabarek, and Roberto Dominguez†
Boston Biomedical Research Institute, 64 Grove Street, Watertown, MA 02472
Edited by James A. Spudich, Stanford University School of Medicine, Stanford, CA, and approved July 28, 2005 (received for review May 10, 2005)
Myosin V is a double-headed molecular motor involved in or-
ganelle transport. Two distinctive features of this motor, proces-
sivity and the ability to take extended linear steps of ?36 nm along
the actin helical track, depend on its unusually long light chain-
binding domain (LCBD). The LCBD of myosin V consists of six
tandem IQ motifs, which constitute the binding sites for calmod-
ulin (CaM) and CaM-like light chains. Here, we report the 2-Å
resolution crystal structure of myosin light chain 1 (Mlc1p) bound
to the IQ2–IQ3 fragment of Myo2p, a myosin V from Saccharomy-
ces cerevisiae. This structure, combined with FRET distance mea-
surements between probes in various CaM–IQ complexes, compar-
ative sequence analysis, and the previously determined structures
of Mlc1p-IQ2 and Mlc1p-IQ4, allowed building a model of the LCBD
of myosin V. The IQs of myosin V are distributed into three pairs.
There appear to be specific cooperative interactions between light
chains within each IQ pair, but little or no interaction between
pairs, providing flexibility at their junctions. The second and third
IQ pairs each present a light chain, whether CaM or a CaM-related
molecule, bound in a noncanonical extended conformation in
which the N-lobe does not interact with the IQ motif. The resulting
free N-lobes may engage in protein–protein interactions. The
extended conformation is characteristic of the single IQ of myosin
VI and is common throughout the myosin superfamily. The model
points to a prominent role of the LCBD in the function, regulation,
and molecular interactions of myosin V.
calmodulin ? IQ motif ? x-ray crystallography ? FRET
of melanosomes and synaptic vesicles in mammals and vacuoles
and mRNA in yeast (1–4). Myosin V is composed of two
identical heavy chains and 12 light chains. Each heavy chain
consists of an N-terminal motor domain, containing the actin-
binding and ATP catalytic sites, followed by the light chain-
binding domain (LCBD), formed by six IQ motifs in tandem, and
the tail domain, composed of regions of coiled-coil and a
globular domain involved in cargo binding. The coiled-coil
regions mediate the association of the heavy chains into dimers.
The IQ motifs are ?25-aa segments, centered around the
consensus sequence IQxxxRGxxxR, and constitute the binding
sites for the light chains, which can be either calmodulin (CaM)
or CaM-related molecules (5, 6).
A number of features distinguish myosin V from other myosin
families. Myosin V has a high duty cycle, defined as the property
to remain attached to actin for a large fraction of the mechano-
chemical cycle (7–9). The high duty cycle of myosin V is
explained by a slow rate of ADP release, which becomes the
rate-limiting step in the ATPase cycle (7). This kinetic adapta-
tion allows myosin V to take multiple steps without dissociating
from the actin filament, that is, myosin V is a processive motor
(10–12). Linked with processivity is the ability of myosin V to
take large steps of ?36 nm (10), a distance equal to the helical
repeat of the actin filament. Such a step size allows myosin V to
walk in a straight line on the actin filament, in a hand-over-hand
fashion (13–16). These characteristics seem to adapt myosin V
for its cellular function, the transport of large cargoes atop the
yosin V is a molecular motor involved in a range of
organelle-transporting functions, including the transport
actin filament while avoiding collisions with cellular structures
(3). Yet, central to this motor’s uniqueness is its unusually long
LCBD (4). A number of laboratories have recently established
a direct connection between the length and structural integrity
of the LCBD and the step size and processivity of myosin V (4,
17–20). These studies focus on the role of the LCBD as a passive
structural device whose function is to amplify small nucleotide-
dependent motions originating in the motor domain, which
explains why this domain is often referred to as the lever arm or
neck domain. By analogy with some class II myosins, for which
the lever arm plays a regulatory function, it is plausible that the
LCBD of myosin V also serves a regulatory role. In agreement
with this view, a number of reports uncover a regulatory effect
of Ca2?and CaM on the conformation and activity of myosin V
that could involve the LCBD directly (21–25). Inhibition of the
ATPase activity of myosin V after increases in Ca2?concentra-
tion has been attributed to the dissociation of CaM from the
heavy chain (26) and?or a conformational change in CaM (27).
Here, we report the results of a comprehensive study of the
structure of the LCBD of Myo2p, a myosin V from Saccharo-
myces cerevisiae (28), including the determination of the 2.0-Å
resolution crystal structure of the tandem IQ repeat IQ2–IQ3 of
Myo2p complexed with two molecules of myosin light chain 1
(Mlc1p), a Myo2p-specific light chain (29), and FRET distance
measurements between probes in various representative IQ–
CaM complexes. The results, combined with the previously
determined structures of complexes of Mlc1p with IQ2 and IQ4
(30, 31) and sequence analysis, allowed building a model of the
LCBD of myosin V. The results have important implications for
our understanding of the structure–function relationship of the
lever arm of myosin V.
Materials and Methods
Preparation of Proteins and Peptides. Mlc1p (UniProt accession no.
P53141), cloned into vector pAED4 under the control of the T7
promoter system, was expressed in Escherichia coli strain BL21
(DE3). The protein was purified by ion exchange chromatography
using a Whatman DE52 column. Human CaM (UniProt accession
no. P62158), cloned into vector pAED4, was expressed by using E.
coli strain BL21 (DE3) and purified on a DEAE-Sephacel column
(Amersham Pharmacia) followed by purification on a Phenyl-
Sepharose column (Amersham Pharmacia). Mutants of Mlc1p
(Ile-64–Met) and CaM (Asp-50–Cys) were generated by using the
QuikChange Site-Directed Mutagenesis Kit (Stratagene). WT
Mlc1p contains a single Met residue at position 109. Mlc1p mutant
Mlc1pIle64Met, containing two Met residues, was made to facilitate
the determination of the structures of Mlc1p–IQ complexes by
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: CaM, calmodulin; LCBD, light chain-binding domain; Mlc1p, myosin light
chain 1; 1,5-IAEDANS, N-iodoacetyl-N?-(5-sulfo-1-naphthyl)ethylenediamine; DABMI,
Data deposition: The atomic coordinates have been deposited in the Protein Data Bank,
www.pdb.org (PDB ID code 1N2D).
*M.T. and G.R. contributed equally to this work.
†To whom correspondence should be addressed. E-mail: email@example.com.
© 2005 by The National Academy of Sciences of the USA
September 6, 2005 ?
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using the anomalous signal from a Se-Met-substituted protein.
CaM mutant CaMAsp50Cyswas designed to allow for labeling with
the FRET acceptor 4-dimethylaminophenylazophenyl-4?-maleim-
ide (DABMI) to monitor the conformation of complexes of CaM
with IQ peptides.
Peptides corresponding to Myo2p (UniProt accession no.
P19524) IQ1 (amino acids 783–805), IQ2 (amino acids 806–
830), IQ3 (amino acids 831–853), IQ4 (amino acids 854–878),
IQ5 (amino acids 879–901), IQ6 (amino acids 902–926), and
IQ2–IQ3 (amino acids 806–853) were synthesized on an Applied
Biosystems 431 peptide synthesizer and then purified by HPLC.
In addition, peptides corresponding to IQ1, IQ3, IQ4, and IQ6,
containing an extra Cys residue at the N terminus (referred to
as Cys-IQ peptides) were synthesized to allow for labeling with
the fluorescent probe N-iodoacetyl-N?-(5-sulfo-1-naphthyl)eth-
ylenediamine (1,5-IAEDANS) (see below). To prevent double
labeling, the Cys residue at position 835 of IQ3 was substituted
with Ser in the Cys-IQ3 peptide. In this way, all of the Cys-IQ
peptides were labeled at structurally equivalent positions, i.e.,
nine amino acids N-terminal to the conserved Ile at position 1
of the canonical IQ motif.
Crystallization, Data Collection, and Structure Determination. The
complex of Mlc1p with IQ2–IQ3 of Myo2p (referred to as
Mlc1p–IQ2,3) was crystallized, and its structure was determined
from the anomalous dispersion signal of a seleno-methionine
derivative as described (31). Refinement was carried out with the
program CNS (32) (Table 1).
Labeling of IQ Peptides and CaM. The Cys-IQ peptides (Cys-IQ1,
Cys-IQ3, Cys-IQ4, and Cys-IQ6) were reduced with 20 mM DTT
for ?30 min. DTT was then removed on a Sephadex G-15
column (Amersham Pharmacia), using 20 mM Hepes, pH 7.5
and 100 mM NaCl as running buffer. The concentration of free
SH groups was determined by the 5,5?-dithiobis(2-nitrobenzoic
acid) method (33), followed by labeling overnight (in the dark,
at 4°C) with 20:1 molar excess 1,5-IAEDANS (Molecular
Probes). Labeled peptides were isolated by HPLC, using a C18
reverse-phase column (Waters), and their concentration was
determined with fluorescamine (34). The purity of the Cys-IQ-
labeled peptides was verified by MS. Labeling of CaMAsp50Cys
with the nonfluorescent FRET acceptor DABMI was carried
out in a similar way. The concentration of labeled CaMAsp50Cys
was determined by Micro BCA assay (Pierce).
Fluorescence Measurements. All fluorescence measurements were
carried out at 20°C in a solution containing 100 mM NaCl, 20
mM Hepes (pH 7.5), and 0.5 mM EGTA. The steady-state
fluorescence emission spectra (?em ? 400–600 nm) were re-
corded on a Cary Eclipse fluorometer (Varian) with excitation
at ?ex ? 337 nm. Fluorescence lifetime measurements were
carried out on a TimeMaster T-4 stroboscope lifetime fluorom-
eter from Photon Technology International, Lawrenceville, NJ.
The scans were collected within a 75-ns window, with 0.5 ns per
channel at ?em ? 500 nm. The concentration of the 1,5-
IAEDANS-labeled IQ peptides was 2 ?M and that of WT CaM
or DABMI-labeled CaMAsp50Cys(when present) was 4 ?M. The
data were fitted with one, two, and three exponentials with
FELIX32 software (Photon Technology International). The dis-
tance R between donor and acceptor was calculated by using
the equation: R ? R0 (?da?(?d ? ?da))1/6, where ?da is the
fluorescence lifetime for a donor–acceptor pair and ?d is the
lifetime for the donor alone (1,5-IAEDANS-labeled IQ peptide
complexed with unlabeled WT CaM). The critical transfer
distance R0was calculated for each peptide from the relation
R0? R0? (?d??d?)1/6, using R0? ? 39.9 Å and ?d? ? 13.5 ns as
reference values for the pair 1,5-IAEDANS–DABMI (35).
Implicit in this calculations is the use of ?2? 2?3, corresponding
to a random orientation of the probes.
Results and Discussion
The Structure of Mlc1p–IQ2,3 and the Relationship Between IQ Se-
quence and Light Chain Conformation.Thestructureofthecomplex
of Mlc1p with IQ2–IQ3 of Myo2p (Mlc1p–IQ2,3) reveals two
Table 1. Refinement statistics
Unit cell parameters
a, b, c, Å
?, ?, ?, °
Resolution range, Å
Free Rfactor, %
80.02, 64.24, 72.79
90.0, 90.0, 90.0
Values in parentheses correspond to last resolution shell. Rfactor? ??Fo?
Fc????Fc?, where Foand Fcare observed and calculated structure factors. Free
the Mlc1p–IQ2 (gray) and Mlc1p–IQ3 (colored as in A) portions of the structure. The side chain of Tyr-843, which forces the opening of the C-lobe in Mlc1p–IQ3,
Structure of Mlc1p–IQ2,3. (A) Ribbon diagram representation of the structure (N-lobes, blue; C-lobes, red; heavy chain, green). (B) Superimposition of
Terrak et al. PNAS ?
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molecules of Mlc1p, one per IQ motif, bound in the canonical
compact conformation (Fig. 1A). Like other members of the
CaM superfamily, Mlc1p presents a dumbbell-shaped structure
with two homologous domains, the N- and C-terminal lobes,
connected by a flexible linker. Each lobe is formed by two
EF-hand-like motifs, each consisting of a pair of ?-helices linked
Ca2?, because of substitutions of some of the amino acids
involved in the coordination of Ca2?in CaM. The compact
conformation characteristic of the two Mlc1p molecules in this
complex has been observed in a number of light chain–IQ
structures (30, 36–39). The N-lobe in these structures is fully
closed (hydrophobic core nonexposed) and interacts with the
C-terminal half of the IQ motif (GxxxR), mainly through elec-
trostatic contacts. The C-lobe assumes a semiopen conforma-
tion, with its hydrophobic core only partially exposed (39), and
interacts with the N-terminal half of the IQ motif (IQxxxR),
primarily through hydrophobic contacts.
Although the complexes are generally similar, important differ-
form Mlc1p–IQ2,3. Because the light chain is the same in both
responsible for these differences. Indeed, other factors, such as
crystal contacts and light chain–light chain interactions across
neighboring IQs, can be ruled out because independently deter-
mined structures of Mlc1p with IQ2 (30) and IQ3 (this 3.0-Å
resolution structure is not described here) are very similar to the
corresponding portions of ternary complex Mlc1p–IQ2,3 (C? rms
deviations of 0.89 and 0.91 Å, respectively).
A better understanding of the relationship between IQ se-
quence and light chain conformation can be obtained from a
superimposition of the Mlc1p–IQ2 and Mlc1p–IQ3 halves of the
structure. Superimposing the IQ portions of these complexes
positions the N-lobes on top of each other, but the C-lobes
diverge significantly (Fig. 1B). A similar observation can be
made of other light chain–IQ complexes (30, 36–39); i.e., a
superposition of the IQ portions of these structures leads to a
good superimposition of the N-lobes, whereas the C-lobes
diverge broadly (data not shown). It appears, therefore, that the
conformational differences observed among compact structures
of light chain–IQ complexes are caused mostly by variations
within the N-terminal IQxxxR portion of the motif, which
interacts with the C-lobe (Fig. 2).
The C-lobe covers approximately three helical turns of the IQ
motif, with the side chains at positions 1 and 5 of the IQ motif
These positions are typically occupied by small, branched ali-
phatic amino acids (Ile, Leu, and Val). However, it is not
uncommon to find bulkier aromatic side chains such as Phe at
position 1 and Phe, Tyr, and Trp at position 5 (Fig. 2). The
presence of these amino acids is typically associated with addi-
tional opening or rearrangement of the C-lobe, as exemplified by
IQ3 of Myo2p, which presents a Tyr residue at position 5.
Because of its polarity, the hydroxyl group of Tyr-843 of IQ3
cannot be accommodated within the hydrophobic core of the
C-lobe, which opens slightly so that the side chain of Tyr-843 can
be directed toward the solvent (Fig. 1B). Similarly, the presence
of a Phe residue at position 1 of the first IQ of smooth muscle
(37) and scallop myosin II (39) leads to local rearrangement of
the C-lobes of the bound essential light chains.
Within the IQxxxR portion of the IQ motif, the strongest
constraint is toward the conservation of the Gln residue at
position 2. In complexes with light chains this amino acid is
buried, forming three strong hydrogen-bonding contacts with
main-chain atoms of the C-lobe. The only other amino acid
known to substitute for Gln at position 2 is Ser (30). Most likely,
the hydroxyl group of Ser coordinates a water molecule that
accounts for some of the hydrogen-bonding contacts of Gln at
position 2. Position 6 of the IQ motif is also highly conserved,
occupied by Arg, and sometimes Lys. The residue at position 7
is also typically involved in hydrogen-bonding contacts with
main-chain atoms of the C-lobe. The remaining amino acids of
the N-terminal part of the IQ motif, positions 3 and 4, are
partially solvent-exposed and vary more widely, with limited
effect on the conformation of the light chain.
Compact and Extended Conformation of CaM–IQ Complexes. Al-
though the compact structures of light chain–IQ complexes
reveal the importance of the N-terminal half of the IQ motif in
determining the conformation of the light chain, departure from
the canonical sequence within the C-terminal GxxxR half of the
motif can have an even greater effect. Thus, substitutions of the
as Arg, Lys, and Met, and?or the loss of the Arg at position 11
result in a noncanonical, extended conformation of the bound
IQ motif is based on the canonical sequence IQxxxRGxxxR. The N-terminal
portion of the IQ motif (IQxxxR), which binds the C-lobe, is colored red. The
C-terminal part (GxxxR), which binds the N-lobe, is colored blue. UniProt
accession numbers are: Myo2p?Sc, P19524; Myo4p?Sc, P32492; MyoVa?Mouse,
Q99104; MyoVa?Rat, Q9QYF3; MyoVa?Human, Q9Y4I1; MyoVa?Chick,
Q02440; MyoVb?Rat, P70569; MyoVb?Human, Q9ULV0; MyoVc?Human,
Q9NQX4; MyoVc?Mouse, Q8BWY8; MyoV?Urchin, Q9NBH3; MyoV?Drome,
O97417; MyoVb?Cele, Q17383; MyoJ?Dicdi, P54697; MyoVI?Human, Q9UM54;
MyoVI?Pig, Q29122; MyoVI?Mouse, Q64331; MyoVI?Chick, Q9I8D1;
Alignment of the LCBD domains of class V and class VI myosins. The
www.pnas.org?cgi?doi?10.1073?pnas.0503899102Terrak et al.
light chain. The extended conformation was originally observed
in the structure of the complex of Mlc1p with IQ4 of Myo2p,
which presents a Lys at position 7 (30). Sedimentation velocity
analysis further demonstrated that the compact complex of
Mlc1p–IQ2 becomes extended when the Gly at position 7 is
replaced by Lys. Conversely, the extended complex of Mlc1p–
IQ4 was converted into a compact complex by a double muta-
tion, which introduced a Gly at position 7 and an Arg at position
11. Note that in this case introducing a Gly at position 7 alone
was insufficient to produce a compact conformation. Based on
these results it was concluded that the presence of a bulky side
chain at position 7 sterically hinders the interaction of the N-lobe
of the light chain with the IQ motif, precluding a compact
conformation (30). Moreover, a compact conformation also
requires the presence of an Arg, or possibly a Lys, at position 11,
as only these two amino acids can form the hydrogen-bonding
interactions with the N-lobe that characterize this conformation.
Based on these results and sequence analysis of the myosin
superfamily (Fig. 2), it can be predicted that the extended
conformation is widespread. For instance, the last IQ motif of
myosin V and the single IQ motif of myosin VI typically present
amino acids at positions 7 and 11 that are consistent with the
extended light chain conformation. However, the most abundant
light chain in myosin V is CaM (1, 21). Will the complexes of
CaM with IQ motifs assume similar compact and extended
conformations as those observed in Mlc1p? To answer this
question, a series of FRET distance measurements between
probes attached to CaM and a representative group of IQ motifs
were carried out.
First, two models, compact and extended, of CaM–IQ com-
plexes were built by using the structures of Mlc1p–IQ2 and
best location for the fluorescence donor and acceptor that,
although sensitive to the conformation of CaM, would not
interfere with the structure of the complexes. Based on this
information, Asp-50 in the N-lobe of CaM was mutated to Cys
(CaMAsp50Cys) and labeled with the acceptor probe DABMI. A
addition of an extra Cys at their N termini: Cys-IQ1, Cys-IQ3,
Cys-IQ4, and Cys-IQ6. The position of the extra Cys is identical
in all four IQ peptides, nine amino acids N-terminal to the first
canonical amino acid of the motif. In this way, the IQ peptides
were labeled at structurally equivalent positions with the fluo-
rescence donor 1,5-IAEDANS. Within this group of IQ motifs,
IQ3 is canonical and should form a compact complex, IQ4 and
IQ6 present noncanonical substitutions at positions 7 and 11 and
are predicted to form extended complexes. IQ1 represents a
group of IQ motifs that present the conserved Arg at position 11,
but have undergone substitutions at position 7 that incorporate
small amino acids such as Ala, Ser, and Thr (Fig. 2). Using the
compact and extended models of CaM–IQ complexes, the
distances between probes attached to these IQs and CaM were
predicted to be ?22 and ?35 Å for the compact and extended
The results of the lifetime distance measurements are in good
agreement with the distances predicted from the models (Table 2)
and indicate that the complexes of CaM–IQ1 and CaM–IQ3 are
compact, whereas those of CaM–IQ4 and CaM–IQ6 are extended.
This finding is evidence that Ca2?-free CaM adopts two different
and extended, which are structurally similar to those in the crystal
is not surprising because despite the evolutionary distance that
separates CaM and the myosin light chains, most of the structural
elements involved in the binding of IQ motifs are conserved (30).
More surprising, however, is the finding that the complex of
CaM–IQ1 is also compact, and possibly even more so than the
complex of CaM–IQ3, despite the fact that the canonical Gly at
position 7 has been replaced by Ala in IQ1. This result allows
refining our understanding of the sequence determinants of the IQ
motif associated with the compact and extended conformations.
Thus, a compact conformation requires the presence of a positively
charged amino acid (Arg or Lys) at position 11 and Gly or a small
amino acid (Ala, Ser, Thr, Val) at position 7 of the IQ motif. On
Arg, Met) and?or the absence of a positively charged amino acid at
position 11 causes the conformation of the bound light chain,
whether CaM or a specific myosin light chain, to be extended.
Light Chain–Light Chain Interactions as a Function of the Spacing
Between IQ Motifs. ThelightchainsinthestructureofMlc1p–IQ2,3
the 14-aa spacing between the last canonical residue of IQ2
(Arg-824) and the first canonical residue of IQ3 (Leu-839), and
IQ2–IQ3 (Fig. 1A). In contrast, in myosin II where the spacing
between IQs is 15 aa, the N-lobe of the essential light chain bound
to IQ1 interacts with the C-lobe of the regulatory light chain bound
to IQ2 (36, 39). This interaction results from a ?30° bend in the
heavy chain segment that separates the two IQs. Such a bend
the constrained structure of the regulatory light chain that binds to
it. Indeed, IQ2 of myosin II lacks the C-terminal GxxxR half, which
is replaced by a second 90° bend in the heavy chain, followed by a
hydrophobic sequence that binds the N-lobe of the regulatory light
chain in a fully open conformation. The direct interaction between
light chains in myosin II has been linked to the Ca2?-mediated
phosphorylation-mediated regulation of smooth muscle myosin. In
myosin V, a spacing of 14 aa between IQ2 and IQ3, and between
IQ4 and IQ5, leads to the lack of stabilizing interactions between
some of the contiguous light chains. Under load the LCBD of
myosin V may bend at the junctions between these IQs. Interest-
ingly, bending of the LCBD of the leading head of myosin V has
Table 2. Fluorescence lifetimes of CaM–IQ complexes
39.921.3 ? 1.7
39.523.4 ? 1.3
39.230.5 ? 0.8
39.3 33.8 ? 1.0
out to minimize ?2. Lifetimes in bold were used to calculate the distances.
Terrak et al.PNAS ?
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vol. 102 ?
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been observed by electron microscopy (13) and has been suggested
to function as a symmetry braking mechanism facilitating the
communication between heads for processive movement (4).
The 14-aa spacing between IQ2 and IQ3, and between IQ4 and
IQ5, effectively separates the LCBD of myosin V into three
semiindependent pairs of IQ motifs: IQ1–IQ2, IQ3–IQ4, and
IQ5–IQ6 (Fig. 2). The spacing between IQs within each pair is only
12 aa. Despite multiple attempts, a structure of a 12-aa-spaced pair
to build by using the structures now available. First, a model was
built for the IQ3–IQ4 pair, based on the structures of Mlc1p–IQ2,3
and Mlc1p–IQ4 (30). A heavy chain fragment corresponding to
IQ3–IQ4 was built by using IQ2–IQ3 as a reference, by turning the
portion of the heavy chain corresponding to IQ4 two amino acids
backward along the ?-helical path. The position of IQ4 thus
Another model was built for IQ1–IQ2, based on the structures of
Mlc1p–IQ2,3 and chicken myosin Va motor–essential light chain
the fact that the models of IQ1–IQ2 and IQ3–Q4 were built
independently, they superimpose well, except for the N-lobe of the
light chain bound to IQ4, which is in the extended conformation.
Although there is no crystal structure of any part of IQ5–IQ6,
the FRET distance measurements performed here indicate that
the light chain bound to IQ6 is in the extended conformation,
and because the spacing between IQ5 and IQ6 is also 12 aa, the
structure of this pair is likely similar to that of the IQ3–IQ4 pair.
V that presents a Pro residue (Pro-896) within its canonical core
(Fig. 2). IQ5 of Myo2p is therefore unique, and unlikely to be
fully functional (see below), which may be connected with the
fact that Myo2p is one of two myosins of class V known to be
nonprocessive (40) (the other being Myo4p). Because our goal
is to build a general model of the LCBD of myosin V, Pro-896
of Myo2p was replaced by Ala, which is the most common amino
acid at this position. A model of IQ5–IQ6 was then obtained by
duplication of the model of IQ3–IQ4 described above.
The models of the three IQ pairs of myosin V reveal a specific
interaction between the light chains that form each pair (Fig. 3).
This interaction involves the first ?-helix of the N-lobe (equiv-
alent to CaM helix A) of the light chain bound to the first IQ and
the second ?-helix of the C-lobe (CaM helix F) of the light chain
bound to the second IQ. The interacting ?-helices run antipa-
rallel for ?1.5 helical turns. The amino acids involved in this
interaction (Glu-15 to Asp-25, Glu-105 to Lys-116 in CaM; and
Asp-9 to Asp-19, Asp-105 to Lys-116 in Mlc1p) are among the
most highly conserved regions between Mlc1p and CaM. Ca2?
binding to the EF-hands of CaM will most likely affect this
interaction. Moreover, because of this interaction, the binding of
light chains to 12-aa-spaced pairs is most likely cooperative.
A Model of the LCBD of Myosin V. The models of the IQ1–IQ2 and
IQ3–IQ4 pairs of Myo2p are naturally connected by the structure
of Mlc1p–IQ2,3. Because the spacing between IQ4 and IQ5 is also
IQ5–IQ6 pair to the first two IQ pairs, yielding a complete model
of the LCBD of myosin V (Fig. 3). However, this model does not
take into account the distribution of CaM and light chains among
the IQ motifs of myosin V. It is generally believed that the LCBD
of myosin V binds between four and five CaMs and one or two
specific light chains (1, 21, 28, 29). Because IQ1 is thought to bind
a light chain (1, 41), CaM is most likely bound to at least four of the
remaining IQs. To check this possibility, the affinities of the six IQs
fluorescence of mutants CaMPhe90Trpand Mlc1pPhe90Trp(data not
shown). Although the measurements indicate a preference of
Mlc1p for IQ2 and CaM for IQ6, the remaining IQs bind CaM and
very low affinities and is unlikely to be fully functional. The affinity
measurements for isolated IQs cannot account for potential coop-
erativity in the binding of light chains to neighboring IQs. Consid-
be subject to regulation and could vary from myosin to myosin, this
study leaves the question of CaM vs. light chain distribution
The model proposed in Fig. 3 should be regarded as a general
model of the LCBD of myosin V and not a specific model of the
LCBD of Myo2p. In addition to the general notion that the LCBD
of myosin V is divided into three semiindependent IQ pairs, the
model reflects the fact that the light chain bound to IQ6 is always
extended, whereas a second extended light chain is associated with
MyoVc?Mouse, and MyoVb?Cele) the second extended light chain
occurs in IQ2, whereas MyoV?Drome is the only myosin of class V
where the second extended light chain occurs in IQ1.
pairs of IQ motifs, with little or no interactions between pairs. The linkers between neighboring IQ pairs are 14 aa long, whereas the linkers between IQs in a
pair are 12 aa long. An enlargement illustrates the interaction between light chains in a pair.
Model of the LCBD of myosin V. The light chains, which can be either CaM or CaM-related molecules such as Mlc1p are colored cyan (N-lobes) and
www.pnas.org?cgi?doi?10.1073?pnas.0503899102Terrak et al.
MyoVc?Human and MyoVc?Mouse are unique in that they present Download full-text
three amino acid inserts in the linker region that separates the first
and second IQ pairs. At least two myosins, MyoVb?Rat and
MyoJ?Dicdi, present three light chains in the extended conforma-
tion and combine two extended light chains in a single IQ pair. As
(Fig. 2), the extended light chain conformation is present in ?35%
of the light chains of myosin V. Interestingly, myosin VI presents a
single IQ motif, which according to its sequence (Fig. 2), is
members of this family. Myosin VI is the only myosin known to
move backward, toward the minus end of the actin filament (42),
and despite a relatively short lever arm, generates large steps and
the question as to whether the extended light chain conformation
plays a role in determining processivity and step size. The crystal
structure of myosin VI, including two CaM molecules, a Ca2?-free
CaM bound to the IQ motif and a Ca2?-loaded CaM bound to a
unique reverse gear insert present in the converter domain of this
myosin, has just been reported (44). However, the CaM molecule
bound to the IQ motif is mostly disordered and its conformation
could not be unambiguously determined.
In summary, the LCBD of myosin V can be conceptually
subdivided into three pairs of IQ motifs. There are extensive
interactions between light chains within pairs, but little or no
for the LCBD to flex under load. Flexing of the LCBD has been
observed by electron microscopy (13) and may play a role in the
communication between heads during processive movement (4).
About 35% of the light chains of myosin V, whether CaM or
CaM-like molecules, are bound in a noncanonical extended
conformation in which the N-lobes do not interact with the IQ
motifs. What is the cellular function of the extended light chain
conformation? The extended light chain conformation could
play a role in myosin localization and?or binding to targets and
effectors. Chicken brain MyoVa, for example, coimmunopre-
cipitates with CaM-dependent protein kinase II (CaMKII), is a
substrate of CaMKII, and activates both the autophosphoryla-
tion and MyoVa phosphorylation activities of CaMKII, possibly
by delivering CaM molecules directly to CaMKII (45).
In class V myosins, the extended light chains are found mainly
within the second and third pairs of IQ motifs, one in each pair.
the single light chain of most myosins VI, are predicted to be
classes, including processivity and their unusually long step size, it
is important to understand the cellular functions of the extended
conformation revealed by this study provide a framework for the
design of in vivo mutants to address this question.
This work was supported by National Institutes of Health Grants
AR46524 and AR41637. Use of the Advanced Photon Source (Argonne,
IL) was supported by the U.S. Department of Energy, Basic Energy
BioCARS facilities at the Argonne National Laboratory (Argonne, IL)
was supported by National Institutes of Health Grant RR07707. Use of
the Cornell High Energy Synchrotron Source (Ithaca, NY) and the
Macromolecular Crystallography Facility at the Cornell High Energy
Synchrotron Source was supported by National Science Foundation
Award DMR 97-13424 and National Institutes of Health Award RR-
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vol. 102 ?
no. 36 ?