From the Department of Biochemistry and Molecular
Biology, the Department of Chemistry, and the Biotechnology
Laboratory, University of British Columbia, Vancouver, British
Columbia V6T 1Z3, the ¶ Plant Biotechnology Institute,
National Research Council of Canada, Saskatoon,
Saskatchewan S7N 0W9, and the
Department of Physiology and
the ** Department of Microbiology and Immunology, University of
Saskatchewan College of Medicine, Saskatoon,
Saskatchewan S7N 5E5, Canada
Received for publication, September 22, 2000
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ABSTRACT |
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The Schizosaccharomyces pombe
Cdc4 protein is required for the formation and function of the
contractile ring, presumably acting as a myosin light chain. By using
NMR spectroscopy, we demonstrate that purified Cdc4p is a monomeric
protein with two structurally independent domains, each exhibiting a
fold reminiscent of the EF-hand class of calcium-binding proteins.
Although Cdc4p has one potentially functional calcium-binding site, it
does not bind calcium in vitro. Three variants of Cdc4p
containing single point mutations responsible for temperature-sensitive
arrest of the cell cycle at cytokinesis (Gly-19 to Glu, Gly-82 to Asp,
and Gly-107 to Ser) were also characterized by NMR and circular
dichroism spectroscopy. In each case, the amino acid substitution only
leads to small perturbations in the conformation of the protein.
Furthermore, thermal unfolding studies indicate that, like wild-type
Cdc4p, the three mutant forms are all extremely stable, remaining
completely folded at temperatures significantly above those causing
failure of cytokinesis in intact cells. Therefore, the altered
phenotype must arise directly from a disruption of the function of
Cdc4p rather than indirectly through a disruption of its overall
structure. Several mutant alleles of Cdc4p also show interallelic
complementation in diploid cells. This phenomenon can be explained if
Cdcp4 has more than one essential function or, alternatively, if two
mutant proteins assemble to form a functional complex. Based on the
structure of Cdc4p, possible models for interallelic complementation
including interactions with partner proteins and the formation of a
myosin complex with Cdc4p fulfilling the role of both an essential and regulatory light chain are proposed.
Cytokinesis begins during anaphase and completes shortly after
mitosis. Prior to this event, a contractile ring containing actin and
myosin forms at the medial plane of the cell. The diameter of this ring
decreases progressively during cytokinesis, presumably due to
contractile forces generated by the myosin motor, and thereby results
in the division of one parent cell into two daughter cells (for review
see Refs. 1 and 2). Although the contractile ring is a complex
structure, it is highly amenable to molecular genetic studies. Well
characterized genes encoding some of the main cytoskeletal components
essential for cytokinesis include tropomyosin (cdc8) (3),
actin (act1) (4), a putative myosin light chain
(cdc4) (5, 6), and two myosins (myo2 and
myp2) (7-9). The product of the myo2 (or
rng5) gene is essential for contractile ring function,
whereas that of the myp2 (or myo3, myo22) gene appears required only under certain conditions
(9). Both Myo2p and Myp2p are type II myosins and thus are predicted to
consist of two heavy chains that dimerize via an extended C-terminal coiled-coil domain. The globular N-terminal domain of each heavy chain
is separated from this coiled-coil by a flexible neck region. In
conventional myosins, such as those found in muscle, two EF-hand proteins, termed the essential light chain
(ELC)1 and regulatory light chain
(RLC), associate with the N-terminal and C-terminal halves of each neck
region, respectively, at IQ-motifs (IQXXXRGXXXR,
where X is any residue) (10, 11). By analogy, we
might also expect an ELC and an RLC to bind each heavy chain in
Schizosaccharomyces pombe, playing a structural support role for the The cdc4 locus, which was identified in the original screens
for cell division control mutants, is required for septum formation and
cell separation (12). Cells with conditionally lethal point mutations
become elongated, dumbbell-shaped, multinucleate and fail to divide
before dying. A similar phenotype is observed when the cdc4
gene is disrupted (5). It was suggested that Cdc4p is a myosin light
chain based on sequence similarities with EF-hand proteins, on
localization to the contractile ring, and on association with Myo2p (5,
13).
Although very likely a myosin light chain, the sequence of Cdc4p is
sufficiently distinct that it is not possible to confidently predict
whether it functions as the structural equivalent of an ELC or RLC.
Site-directed mutation of a conserved residue in the IQ motif proximal
to the Myo2p N terminus results in loss of association with Cdc4p in
immunoprecipitation assays (13). This indicates that Cdc4p associates
with Myo2p at least at the equivalent of an ELC-binding site (14).
However, S. pombe cells expressing only the mutated form
of Myo2p were still viable, suggesting that some binding of Cdc4p may
occur in vivo (13). Alternatively, Cdc4p may also interact
with other proteins in the contractile ring, besides Myo2p, such as
Myp2p or Rng2p (7, 9, 15). Recently, a gene that presumably encodes a
small EF-hand protein was annotated as a putative RLC by the Sanger
Centre Genome Project (accession number CAB54151). This RLC-like
protein also localizes to the contractile ring and appears to interact
with Myo2p at the second IQ motif, a recognized binding site for RLC in
conventional myosins.2 Thus,
current evidence suggests that Myo2p is a conventional myosin with
Cdc4p serving as an ELC and possibly a second related protein as an
RLC.
However, the report that initially identified the cdc4 locus
also described a pair of closely linked cdc A major goal of our research is to understand the structural basis for
the essential functions of Cdc4p, with particular emphasis on defining
how conditional mutants of Cdc4p fail to complete cell division under
restrictive conditions and on explaining the observed interallelic
complementation of these mutants. There are currently no structural
data available for any of the cytoskeletal proteins involved in the
mechanism and regulation of cytokinesis. To this end, we have used NMR
spectroscopy to determine the tertiary structure of wild-type Cdc4p and
to characterize three temperature-sensitive mutants of this protein. In
parallel with the accompanying paper (49) describing the identification
by genetic and immunochemical methods of proteins that interact with
Cdc4p, we discuss possible models for the function of this essential
component of the S. pombe cytokinesis machinery.
Labeling and Purification of Cdc4p for NMR Spectroscopy--
A
cdc4 cDNA construct in the Escherichia coli
expression vector pRSET B (Invitrogen Corp., San Diego, CA) was a gift
of Dr. Dan McCollum (Vanderbilt University). Vectors for expression of cdc4 temperature-sensitive mutants were made by cloning
polymerase chain reaction-amplified coding regions of
cdc4
E. coli strains BL21( NMR Spectroscopy--
NMR experiments were performed on ~5
mM wild-type or mutant Cdc4p dissolved in 100 mM KCl, 1 mM EDTA, 8 mM
dithiothreitol, pH 6.5, in either 90% H2O, 10%
D2O or 100% D2O. All data were recorded at
30 °C with a Varian Unity 500-MHz NMR spectrometer equipped with a
pulsed-field gradient accessory. NMR data were processed using NMR pipe
(19) and analyzed using the program PIPP (20). Essentially complete
1H, 13C, and 15N spectral
assignment of wild-type Cdc4p was obtained using an extensive set of
gradient-enhanced three-dimensional experiments as outlined in Ref. 21.
The diastereotopic methyl groups of valine and leucine were
stereospecifically assigned using biosynthetically directed
13C labeling (22). Both histidine residues in Cdc4p were
shown to be partially deprotonated and predominantly in the
N
Three-dimensional structures were computed from experimental restraints
starting with an extended chain using a simulated annealing protocol
with X-PLOR version 3.8 (25). Experimental distance restraints were
obtained as described (26-28) using three-dimensional 15N-NOESY HSQC, three-dimensional simultaneous
13C/15N NOESY-HSQC, four-dimensional
13C/13C HMQC-NOESY-HMQC spectra, and a
two-dimensional homonuclear NOESY experiment in D2O (for
NOEs involving aromatic protons), all recorded with a
Circular Dichroism Spectroscopy--
The circular dichroism
spectra of ~10 µM wild-type or mutant Cdc4p in 30 mM potassium phosphate buffer, pH 6.5, were acquired with a
Jasco J-730 CD spectropolarimeter using a 0.1-cm water-jacketed quartz
cell. Thermal denaturation curves were recorded by monitoring the
signal at 222 nm as a function temperature, increased at
1 °C/min.
Cdc4p Is Made of Two Distinct Domains Joined by a Flexible
Linker Region
The tertiary structure of Cdc4p was determined from 2195 NMR-derived restraints using a simulated
annealing protocol (Fig. 1A).3
The ensemble of 26 calculated structures exhibit good covalent geometry
as indicated by low r.m.s. deviations from idealized values and by low
NOE, dihedral angle, and van der Waals energies (Table
I). For all 26 structures, 99.3% of the main
chain (
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
-helical neck region of myosin.
mutants that complemented one another and recombined with low frequency
(12). This interallelic complementation, which cannot be explained if
the only role of Cdc4p is to serve as an ELC, might arise if Cdc4p has
two independent, essential functions, each selectively disrupted by a
single mutation. Indeed, as shown in the accompanying paper (49), Cdc4p
interacts with several additional proteins besides Myo2p, including a
putative phosphatidylinositol 4-kinase. Alternatively, interallelic
complementation can occur if two mutant forms of a protein assemble to
form a functional oligomeric complex in a diploid cell. In support of
this latter case, an attractive structural model can be developed in
which two mutant forms of Cdc4p, serving as both an ELC and RLC, bind to myosin at both IQ motifs and physically interact with one another via their respective wild-type surfaces.
MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
G19E, cdc4
G82D, and
cdc4
G107S into pT7-7 (16). All sequences were
confirmed using a model 370A automated sequencer (PE Applied Biosystems Inc.).
DE3) (Stratagene, La Jolla, CA) and
CT19 (avtA::Tn5/trpB83::Tn10/dcm ompT
lon 8DE3 ilvE12 tyrB507 aspC13) (17) were transformed with the
appropriate expression vector immediately prior to use. Unlabeled,
uniformly 15N- and 13C-labeled and selectively
-15N-labeled proteins were prepared using media as
described previously (18). After induction with 1 mM
isopropyl-1-thio-
-D-galactopyranoside and growth
overnight at 25-30 °C, E. coli cells were harvested by
centrifugation and suspended in 30 ml of Buffer A (30 mM
Tris-HCl, pH 7.5, 2 mM 2-mercaptoethanol, 15% glycerol)
per 500 ml of culture. Cells were lysed using a French pressure cell,
followed by centrifugation. The supernatant was loaded onto a Fractogel
anion exchange column (Merck), which was developed at 3 ml/min in
Buffer A using a linear NaCl gradient from 0 to 600 mM.
SDS-polyacrylamide gel electrophoresis analysis showed that Cdc4p
eluted from the column between 300 and 400 mM NaCl. Protein
from these fractions was precipitated with
(NH4)2SO4 (70% saturation) and
dissolved in Buffer A containing 150 mM NaCl. The protein
solution was loaded onto a Superdex 75 gel filtration column (Amersham
Pharmacia Biotech), and fractions were collected at 1 ml/min. The Cdc4p
was precipitated with (NH4)2SO4 (70% saturation), dissolved in 25 mM
NH4HCO3, pH 8.5, and loaded on a Superdex 75 column equilibrated with 25 mM
NH4HCO3 for buffer exchange. The final
fractions were pooled, concentrated in Centricon-3 microconcentration
device (Amicon Inc., Beverly, MA), and lyophilized in a Speed-Vac. The
identity of each protein was confirmed by mass spectrometry. During
preparation of the protein, the N-terminal methionine was removed by
E. coli proteases.
2H tautomeric form at pH 6.5 using the HMBC
experiment (23). Assignments of the 1HN and
15N resonances in uniformly 15N-labeled
Cdc4p-G19E, Cdc4p-G82D, and Cdc4p-G107S were obtained using
three-dimensional 15N-TOCSY/NOESY-HSQC experiments. To
confirm these assignments, HSQC spectra were also recorded on
Cdc4p-G107S selectively labeled with 15N-Phe and
15N-Leu, and on Cdc4p-G82D selectively labeled with
15N-Phe, 15N-Leu, 15N-Val, and
15N-Ala. 15N T1,
T2, and heteronuclear
15N{1H}-NOE relaxation data were recorded
and analyzed on uniformly 15N-labeled wild-type and mutant
proteins, as described (24).
mix = 75 ms. Hydrogen bonds were included as distance restraints for those amides remaining protonated 60 min after transfer
of the protein into D2O buffer. Torsion angle
restraints were obtained from an analysis of an HNHA experiment (29)
and
restraints from an analysis of the
dN
/d
N ratio according to
(30).
1 angles were restrained according to a staggered rotomer
model using coupling patterns observed for stereospecifically assigned
H
,
' in 15N-TOCSY-HSQC and HNHB spectra and for the
methyls of The, Ile, and Val in long range
13C
-15N and -13C' correlation
spectra (29).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
,
) angles fall in the core or allowed regions of the
Ramachandran map, as determined using PROCHECK-NMR (31). With the
exception of the N and C termini (residues 2-7 and 138-141,
respectively) and a central linker region (residues 65-78), the
structures of the backbone and core side chain atoms of Cdc4p are well
defined by NMR data.
View larger version (62K):
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Fig. 1.
Cdc4p is composed of two structurally
distinct domains connected by a flexible linker. Shown is the
ensemble of 26 structures calculated for the wild-type protein,
superimposed using the backbone atoms in the -helices of the
N-terminal domain (A, all residues; B, residues
2-66 only), and the C-terminal domain (C, residue 77-141
only). Due to the flexible linker, the N- and C-terminal domains do not
have a fixed orientation with respect to one another. A MOLSCRIPT
ribbon diagram of one representative structure of Cdc4p is shown in
D, with
-helices colored as in A-C and
-strands indicated as white arrows. Helix boundaries are
as follows: A (8-14), red, B (26-35), orange,
C (41-49), yellow, D (58-64), green,
E (79-86), green, F (96-105), blue,
G (113-119), purple, and H (133-137), magenta.
The short anti-parallel
-sheets encompass residues 22-24 and 54-56
in the N-terminal domain and 93-95 and 127-129 in the C-terminal
domain. Also indicated in D are the positions of point
mutations in the N- (F12L, G19E, and R33K) and C-terminal domains
(F79S, G82D, and G107S) causing temperature-dependent cell
growth arrest at cytokinesis. Serines 2 and 6, which are sites of
phosphorylation in vivo (40), lie at the exposed N terminus
of the protein.
Structural statistics
As shown in Fig. 1D, Cdc4p adopts a dumbbell-shaped
structure with distinct N and C domains. Each consists of four
-helices, named A through D in the N domain (Fig. 1B) and
E through H in the C domain (Fig. 1C), and a short
two-stranded anti-parallel
-sheet. Joining the two domains is a
proline-rich linker region (residues 66-77) that is devoid of any
regular structure. Based on 15N NMR relaxation measurements
(see below), the linker is highly flexible in solution, and thus the N-
and C-terminal domains do not have a fixed orientation with respect to
one another. Consistent with this conclusion, Cdc4p can be chemically
cleaved into two separate domains, and each domain can be expressed in
isolation (49). The monomeric nature of Cdc4p is indicated by the
narrow resonances detected within its NMR spectra and confirmed by both heteronuclear relaxation measurements, which reveal 15N
T1 and T2 values
consistent with a protein of ~15 kDa total molecular mass, and
by equilibrium ultracentrifugation runs which indicated an average
molecular mass of 14.1 ± 0.9 kDa over three iterations at 15,000 rpm (not shown).
Cdc4p Contains Four EF-hand Motifs but Does Not Bind Calcium
Consistent with predictions based on sequence alignments, Cdc4p
contains four EF-hand structural motifs (32), defined by helices A/B
and C/D in the N-terminal domain (Fig. 1B) and by E/F and
G/H in the C-terminal domain (Fig. 1C). The two EF-hands within each domain are joined by the antiparallel pairing of the short
-strands located within the loop regions between their constituent helices.
The structural similarity between Cdc4p and other EF-hand proteins such as calmodulin prompted us to investigate the possibility of calcium binding by Cdc4p. Accordingly, 1H-15N HSQC NMR spectra of Cdc4p were recorded both in the presence of excess calcium and of excess EDTA. The NMR spectrum of a protein is extremely sensitive to structural and electrostatic perturbations and thus serves as an excellent indicator of ligand binding. The complete lack of any significant spectral changes under either condition (data not shown) strongly suggests that Cdc4p does not bind calcium with any appreciable affinity.
Dynamic Properties of Cdc4p
15N NMR relaxation measurements were carried out to
investigate the dynamic properties of the backbone of Cdc4p. As shown
in Fig. 2, residues at the N and C termini
and within the linker region connecting the two domains of the protein
exhibit long transverse (T2) relaxation times
and reduced heteronuclear 15N{1H}-NOE
values compared with those in regions of well defined secondary structure. These results are diagnostic of fast internal motions relative to the global tumbling of the protein (33). Therefore, the
apparent disorder or high r.m.s. deviations seen for these regions
within the ensemble of structures calculated for Cdc4p (Fig.
1A) can be attributed to conformational flexibility on the sub-nanosecond time scale. The dynamic disorder of the termini and
linker are further supported by the observation that backbone amides
within these regions do not show any significant protection from
hydrogen-deuterium exchange with solvent (data not shown). Together,
these measurements confirm that the N- and C-domains of Cdc4p are
independent structural units tethered by a flexible proline-rich linker
sequence.
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15N NMR relaxation measurements also indicate the loop regions in Cdc4p are flexible on a sub-nanosecond time scale. Slightly longer transverse (T2) relaxation times and reduced heteronuclear 15N{1H}-NOE values for amides in the loop regions of the EF-hands I, III, and IV (residues 16-19, 87-90, and 121-126), as well as the loop regions between the EF-hands in the two domains (residues 36-40 and 106-112), indicate more mobility than seen for amides forming elements of regular secondary structure. This corresponds to the slightly higher r.m.s. deviations observed for these loop residues in the ensemble of calculated structure (Fig. 1, B and C). Not surprisingly, the dynamic properties of the backbone of Cdc4p are similar to that observed for other EF-hand proteins, such as apo-cTnC (34).
It is interesting to note that, although both domains of Cdc4p are of similar size, the backbone root mean square deviations from the average structure for the C-domain are almost twice that of the N-domain (Table I). This is a direct result of the measurement of fewer dihedral and medium/long range interproton distance restraints in the C-domain versus the N-domain. However, we attribute this to different motional properties of the domains of Cdc4p rather than poorer spectral resolution for residues in the C-domain. This conclusion is based on two related observations. First, peaks corresponding to several residues in the C-domain in the 1H-15N-HSQC NMR spectrum of Cdc4p exhibit weak intensity (Gly-82, Gln-84, Val-85, Phe-86, Met-93, Ile-94, Gly-95, Asn-112, Asp-125, Val-129, Tyr-131, and His-132), suggestive of a conformational exchange phenomenon occurring on a sub-millisecond time scale. Second, based on 15N NMR relaxation measurements, the average 15N T1 and T2 values for the N-domain (residues 8-64) are 557 ± 27 and 87.8 ± 7.3 ms, respectively, whereas those for the C-domain (residues 79-137) are 566 ± 26 and 75.6 ± 16.5 ms, respectively (Fig. 2). In particular, the shorter T2 values, along with a greater deviation from the average, for amides in the C-domain of Cdc4p also suggest that the backbone of this portion of the protein may undergo slow motions that could lead to line broadening. In addition, the average heteronuclear 15N{1H}-NOE values for the N- and C-domains are 0.67 ± 0.06 and 0.58 ± 0.09, respectively. The slightly lower NOE values for amides in the C-domain suggest that the residues may also be under rapid internal motions on a sub-nanosecond time scale. A similar pattern of complex relaxation behavior was observed in a 15N NMR relaxation study of calmodulin (35).
Structural and Dynamic Properties of Cdc4p Temperature-sensitive Mutants
Three conditionally lethal, loss-of-function point mutations in
Cdc4p were studied using 1H-15N NMR
spectroscopy. The positions of these mutations (G19E, G82D, and G107S)
are mapped on the structure of the wild-type protein in Fig.
1D. To investigate the structural and dynamic effects of
these amino acid substitutions, three approaches were taken. First,
1HN and 15N chemical shift
differences between the wild-type and mutant proteins were measured as
a sensitive indicator of structural perturbations (Fig.
3). Second,
3JHN-H coupling constants were
measured to investigate possible changes in backbone
dihedral
angles. Finally, 15N relaxation measurements were utilized
to probe changes in the dynamic behavior of the amide residues in the
backbone of the protein. As discussed below, these measurements
demonstrated that each of the mutant forms of Cdc4p adopts a stable,
folded native-like conformation at pH 6.5 and 30 °C, with only small
structural and dynamic perturbations occurring within the domain of the
Cdc4p containing the site of substitution.
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Cdc4p-G19E--
This glycine to glutamic acid mutation
occurs in the exposed loop region of the first EF-hand (Fig.
1D). Although the wild-type (,
) angles would disfavor
a non-glycine residue, any structural perturbations resulting from the
substitution appear small as evident by the localized chemical shift
changes (Fig. 3) and the lack of significant changes in the
3JHN-H
coupling constants. It is
interesting to note that the region of the wild-type protein near
position 19 has relatively high root mean square deviations in
the ensemble of calculated structures (Fig. 1), as well as anomalous
15N T2 and heteronuclear NOE values
(Fig. 2), both of which are indicative of local conformational
mobility. The 15N relaxation parameters of Cdc4p-G19E also
remained similar to those of the wild-type protein. Therefore, it is
likely that Cdc4p readily accommodates a glutamic acid residue at this
surface position without significantly altering its structure or
dynamic properties.
Cdc4p-G82D--
This glycine to aspartic acid mutation falls
within the middle of helix E (Fig. 1D). The chemical shifts
of amides throughout the linker and the entire C-domain are perturbed,
suggesting that small structural changes occur throughout the second
half of the protein in response to the amino acid substitution.
Furthermore, based on changes in the measured
3JHN-H, coupling constants for
residue 87 at the C terminus of helix E (10 to 4 Hz) and residue 96 at
the N terminus of helix F (2 to 10 Hz), it appears that the backbone
angles have shifted such that helix E is lengthened and helix F
shortened. It is likely that these structural changes, which may
reflect a slight rotation of helix E, are necessary to displace the
aspartic acid side chain from the hydrophobic interface between helices
E and F. Relaxation measurements also reveal that the 15N
T2 lifetimes of residues 72, 77, and 78 are
increased as compared with wild-type, indicating greater flexibility in
the linker region of the G82D mutant.
Cdc4p-G107S--
This glycine to serine mutation occurs within the
exposed loop between the 2 EF-hands of the C-domain of Cdc4p (Fig.
1D). Chemical shift perturbations due to the amino acid
substitution are most pronounced for amides near the site of the
mutation and smaller throughout the rest of the C-domain. In contrast,
no significant differences in
3JHN-H coupling constants or
15N relaxation parameters were detected between the
wild-type and mutant protein (Fig. 3). Together, these data suggest
that subtle structural perturbations arise throughout the C-domain in
response to the amino acid substitution. These perturbations may result from a change in the conformation of the loop region due the
introduction of a side chain at position 107 and thus an alteration in
the packing of helices G and H.
Folding Studies of Cdc4p Temperature-sensitive Mutants
NMR measurements clearly reveal the three Cdc4p mutants adopt a
folded, native-like structure. Therefore, the temperature-sensitive phenotypes associated with each mutation are unlikely to result from a
significant change in the tertiary structure of the protein. This is
consistent with cell viability under permissive conditions. To
investigate the possibility that the temperature-sensitive phenotypes
arise from destabilization of Cdc4p, circular dichroism spectroscopy
was used to monitor the thermal unfolding transitions of the purified
Cdc4p variants (data not shown). Cdc4p as well as Cdc4p-G19E, -G82D,
and -G107S are all extremely stable, remaining completely folded at
>70 °C. To obtain measurable unfolding transitions, 4 M
urea was added as a denaturant to the sample buffer. Under these
conditions, all four proteins exhibited broad, biphasic thermal
denaturation curves, suggesting that the N- and C-domains of Cdc4p
unfolded independently and at different temperatures. Wild-type Cdc4p
and both Cdc4p-G82D and -G107S exhibited apparent mid-point unfolding
temperatures of ~50 °C in 4 M urea at pH 7.0, whereas
that of Cdc4p-G19E was reduced to ~41 °C. Further deconvolution of
the data to yield distinct denaturation transitions was not feasible.
Regardless, these results strongly suggest that each of the three Cdc4p
mutants adopts very stable, native-like folded structures and that the
temperature-sensitive phenotypes must arise directly from a disruption
of the function of Cdc4p, rather than indirectly through a disruption
of its overall structure.
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DISCUSSION |
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Cdc4p Is a Two Domain, EF-hand Protein That Does Not Bind Calcium-- Cdc4p is the first component of the S. pombe contractile ring to have its tertiary structure determined. As expected from sequence comparisons (5), the basic fold of Cdc4p is that of a dumbbell-shaped protein with two independent domains connected by a flexible linker. Although each domain contains two EF-hand motifs, only the third has the appropriate side chains for metal chelation (36). However, Cdc4p does not bind calcium in vitro as evident by the lack of any NMR spectral changes upon addition of excess CaCl2 or EDTA. Similarly, the essential light chain (ELC) from chicken skeletal muscle has only one potential metal-binding site (EF-hand 3) but also does not bind this metal ion (10, 37). As summarized in Table II, Cdc4p differs in its precise tertiary structure from several well characterized EF-hand proteins, such as calmodulin, troponin C, and the ELC or RLC bound to myosin. These differences undoubtedly reflect the diversity of functions served by this large family of structural and regulatory proteins (for review see Refs. 38 and 39).
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Cdc4p as an Essential Light Chain-- Structural, biochemical, and genetic evidence suggest strongly that a primary function of Cdc4p is that of an essential light chain. This evidence includes the following: (i) similarity of sequence, structure, and lack of calcium binding with well characterized ELCs; (ii) direct interaction with the contractile ring myosin, Myo2p, as demonstrated by coimmunoprecipitation assays (13) and immunosorbent assays (49); and (iii) disruption of this interaction in vitro by mutation of the first IQ motif of Myo2p (13), which corresponds to an established ELC-binding site in conventional myosins.
By analogy to the structure of a scallop muscle ELC-RLC-myosin heavy chain headpiece ternary complex determined by x-ray crystallography (37, 41, 42), the C-terminal domain of Cdc4p most likely interacts with the first IQ motif in Myo2p. However, as indicated in Table II, the interhelical angles within the EF-hand motifs of free Cdc4p differ from those observed for the muscle ELC bound to myosin. In particular, in the crystalline scallop ternary complex, the ELC is anchored tightly to the myosin headpiece via its C-terminal domain, which adopts a "semi-open" conformation, and more weakly via its N-terminal domain in a "closed" conformation (10, 41). (The "open" conformation for EF-hand proteins is defined by an interhelical angle close to 90° (perpendicular), whereas the closed conformation is characterized by an interhelical angle closer to 180° (anti-parallel) (38)) In contrast, both N- and C-domains of Cdc4p in solution exhibit rather mixed conformations, reminiscent of that of calcium-loaded cTnC. This comparison implies that Cdc4p must undergo a conformational rearrangement upon binding to Myo2p. Such conformational changes are well characterized for the association of many EF-hand proteins with their target polypeptides and often serve as a mechanism for the regulation of their activities, e.g. via the binding of ligands such as calcium. Dynamic studies of Cdc4p by 15N relaxation suggests that, in particular, its C-terminal domain exhibits conformational mobility on a sub-millisecond time scale and thus should readily undergo any required structural rearrangements upon binding Myo2p. Interestingly, in contrast to the light chains from chicken smooth muscle myosin that are largely insoluble and tend to aggregate in the absence of myosin,4 Cdc4p is very soluble and remains monomeric at relatively high concentrations. This may reflect different functional requirements for the ELC in cytokinesis and in muscle contraction. Regardless, the structure of Cdc4p represents the first example of an isolated ELC and, in combination with that the scallop ELC in a crystalline ternary complex, provides a view of the free and bound conformations of the essential myosin light chain.
In the absence of direct structural information on Cdc4p bound to its
target sequence(s) in Myo2p, the scallop ELC-RLC-myosin head piece
complex reveals possible explanations for the conditionally lethal
phenotypes resulting from several point mutations in this S. pombe protein. For example, the temperature sensitivity of Cdc4p-G82D may arise from disruption of the expected shallow
hydrophobic pocket that is predicted to be generated in the bound
semi-open conformation. Although this residue is not well conserved
among ELCs, it always contains a nonpolar side chain. As indicated by the NMR characterization of Cdc4p-G82D, an aspartic acid residue at
position 82 may result in a disruption of the packing between helices E
and F and thus lead to a subtle alteration in the overall structure of
the C-terminal domain. As the temperature is increased, this could
destabilize the binding of Cdc4p to Myo2p and thereby disrupt
cytokinesis. In a similar manner, the G107S mutation would alter the
docking of the C-terminal domain of Cdc4p onto an IQ motif, as the
residues in the linker between the two EF-hands (including position
107) are important for this binding event (42). In contrast, the G19E
mutation may not have a direct effect on the binding of Cdc4p to the IQ
motif but rather could affect the stabilization of the ternary myosin
complex involving Cdc4p in the ELC position and a second protein in the
RLC position. This hypothesis derives from the observation that the
region in the scallop ELC equivalent to Gly-19 in yeast Cdc4p is
important for inter-light chain hydrogen bonding (42) (Fig.
4).
|
Cdc4p Interacts with Several Protein Partners-- The early observation by Nurse et al. (12) that conditionally lethal alleles cdc4-G19E and cdc4-G107S complemented one another under restrictive temperatures in diploid cells has recently been extended to include the combinations of cdc4-F12L/cdc4-G82D and cdc4-F12L/cdc4-R33K (49). This interallelic complementation cannot be easily rationalized if the only role of Cdc4p is that of an ELC. One possible explanation is that Cdc4p may have two or more essential and independent functions, each being selectively disrupted by a mutation. Consistent with this possibility, Cdc4p appears to bind several proteins besides Myo2p. For example, in myo2-null mutants, Cdc4p is still recruited to the contractile ring, perhaps by associating with Myp2p, a second myosin heavy chain found in S. pombe. Furthermore, a synthetic lethal genetic interaction between cdc4 and rng2, a gene encoding a contractile ring protein similar to human IQGAP1, has been documented (15). This latter protein is known to bind actin and calmodulin, as well as to regulate Rho GTPases (15). Phenotypic analysis has led to the suggestion that Cdc4p and Rng2p may be involved in organizing actin cables into rings (43).
As presented in the accompanying paper (49), using a yeast two-hybrid
screen, we have identified two additional Cdc4p-binding proteins not
obviously associated with cytokinesis. The first of these is a putative
phosphatidylinositol 4-kinase (PI 4-kinase). This interaction is
dependent only on the C-terminal domain of Cdc4p and is disrupted by
the mutation G017S but not F79S or G82D. This implicates residues near
position 107 as mediating association with the kinase. Note, however,
that viability in cdc4 strains can be restored
by exogenous expression of full-length Cdc4p but not by either its
isolated N- or C-terminal domain. Thus, although the interaction of
Cdcp4 with PI 4-kinase occurs via the C-terminal domain alone, the
attached N-terminal domain may be required for biological function,
perhaps by mediating the formation of higher order protein assemblies.
The second Cdc4p-interacting partner identified by two-hybrid screening
is similar to S. cerevisiae Vps27p, a protein implicated in
vacuolar and endocytic membrane traffic. In contrast to the case of the
PI 4-kinase, this interaction is dependent on both domains of Cdc4p and
is selectively disrupted by the substitution of F12L. Position 12 is
located within the hydrophobic core of Cdc4p, and thus this mutation
may perturb, albeit subtly, the structure of its entire N-terminal
domain. Possible biological implications of these two partnerships are discussed in the accompanying paper (49). However, it remains to be
established which, if any, of these interactions represent essential
functions of Cdc4p in S. pombe and thus could account for
the observed phenomenon of interallelic complementation.
A Structural Model How Cdc4p May Interact with Myosin as Both an ELC and RLC-- A second mechanism by which interallelic complementation may arise is through the assembly of mutant forms of a protein into a functional complex in diploid cells. In Fig. 4, we propose a model in which two Cdc4p molecules could bind to the neck region of each Myo2p heavy chain, in a fashion structurally equivalent to the ternary complex involving an ELC, RLC, and the headpiece of scallop muscle myosin heavy chain (37, 41, 42). The main features of the model are as follows: (i) one Cdc4p interacts with Myo2p as the structural equivalent to an ELC bound to the myosin heavy chain; (ii) a second Cdc4p interacts with Myo2p as the structural equivalent of an RLC bound to muscle myosin; (iii) the N-domain of Cdc4p (in an ELC-like position) interacts with the C-domain of Cdc4p (in an RLC-like position); (iv) both Cdc4p-Cdc4p and Cdc4p-Myo2p interactions are essential for function.
The key feature of this model is that Cdc4p associates with Myo2p at both the ELC- and RLC-binding sites of the myosin heavy chain. That Cdc4p binds to the first IQ motif of Myo2p was shown in Ref. 13 as mutation of Arg-770 within this motif abolished interactions in coimmunoprecipitation assays. In conventional myosins, the first of two IQ motifs is a binding site for an ELC. Our model suggests that a second Cdc4p could also occupy the RLC-binding site in Myo2p. This hypothesis is based on the following: (i) the NMR-derived structure of Cdc4p, combined with dynamic measurements, indicating that this protein exhibits flexibility and thus could conceivably undergo changes in conformation to resemble those of either the bound ELC or RLC (Table II); (ii) NMR and CD spectroscopic studies of three mutant forms of Cdc4p which reveal that the temperature-sensitive defects in cytokinesis are caused by disruption of function rather than of overall structure; (iii) interallelic complementation that is readily explained if a ternary complex comprising two Cdc4p is formed with myosin; (iv) the demonstration that the two domains of Cdc4p are not functionally independent as only the intact full-length protein can rescue mutant phenotypes (49); and (v) analogy to the crystallographic structure of the scallop RLC-ELC-myosin ternary complex, in which important interactions exist not only between each light chain and myosin but also between the C-domain of the ELC and the N-domain of the RLC (42).
It should be noted that three exhaustive genetic screens for
cdc mutants failed to identify another protein
that could serve as an RLC (12, 43, 44). In Dictyostelium,
mutations in either its ELC or RLC cause cytokinesis defects and loss
of viability (45-48), suggesting that such a protein should have been
detected if essential for cell division. However, a gene that
presumably encodes a small EF-hand protein was recently annotated as an
RLC based on sequence similarity (33.9% identity in 171 amino acids) to Drosophila melanogaster myosin RLC (Sanger Center
S. pombe sequencing group, accession number CAB54151). This
RLC-like protein also localizes to the contractile ring and appears to interact with Myo2p at the second IQ motif, a recognized binding site
for RLC in conventional myosins.2 Since the gene encoding
this protein was not detected as a cdc
mutant,
it remains to be established if it plays an essential role in
cytokinesis. It is also possible that Cdc4p and the RLC-like protein
may bind to myosin interchangeably or at different times during the
formation or function of the contractile ring, allowing for the
formation of the postulated Cdc4p-Cdc4p-Myo2p complex.
In the model of Fig. 4, both domains of Cdc4p are required to associate with Myo2p. By analogy to the scallop RLC-ELC-myosin ternary complex, the binding of Cdc4p in the RLC position is required to stabilize the weak binding of the Cdc4p N-domain in the ELC position. This is consistent with the experimental observations that the two domains of Cdc4p are not functionally independent and that expression of intact Cdc4p is required to rescue the cytokinesis defects observed in yeast with mutant alleles of Cdc4p. For example, expression of full-length wild-type Cdc4p in cells bearing mutations in the N-domain (G19E) or C-domain (G107S) restored viability at the restrictive temperature, whereas expression of the N-domain or C-domain alone or in combination did not (49). These results are similar to those observed in Dictyostelium where expression of an ELC with deletions in either the N terminus or C terminus abolished binding to myosin and failed to rescue cytokinesis defect of ELCnull cells (47). The fact that free Cdc4p is monomeric in solution indicates that, as with muscle ELC and RLC, the postulated interactions are weak, occurring only when both light chains are bound to the myosin heavy chain.
Within the context of the proposed model, the structure of Cdc4p provides a rationale to explain how single point mutations cause temperature-dependent failure of cytokinesis. A key feature of the model is that two Cdc4p interacts in different ways with the heavy chain of Myo2p depending upon their position as the structural equivalents to an ELC and RLC. Thus, a single point mutation may have a destabilizing effect when Cdc4p is in one position but not in the other, providing a rationale to explain the observed cases of interallelic complementation. For instance, position 107 occurs in a highly conserved region of Cdc4p and the glycine to serine substitution causes subtle structural perturbations throughout its C-terminal domain. These perturbations may interfere with potential hydrogen bonding between the glutamine side chain in position 2 of the second heavy chain IQ motif and the carbonyls of Leu-107 and Glu-108 of Cdc4p, thereby weakening the binding of Cdc4p-G017S to Myo2p. Furthermore, in the scallop muscle RLC-ELC-myosin ternary complex, the equivalent residues to Gly-107 of Cdc4p in the RLC position (Fig. 4, red) hydrogen-bonds via its backbone amide and carbonyl atoms to the backbone amide and carbonyls of the equivalent residues to Arg-17 and Gly-21 of Cdc4p in the ELC position (Fig. 4, blue). Strikingly, these latter residues surround the G19E mutation in the N-domain of Cdc4p. Thus, in addition to disrupting direct interactions with Myo2p, mutation of either residue 19 or 107 may destabilize the association of two Cdc4p molecules acting as the structural equivalents of the ELC and RLC. Since interaction between the two light chains is required for ternary complex formation, the mutations would lead to a conditionally lethal phenotype. Extending this argument, the fact that diploid cells expressing G107S and G19E mutant alleles are viable at the restrictive temperature can be explained with G107S mutant protein in the ELC position and G19E in the RLC position. This would preserve an intact wild-type interface between the two Cdc4p (Fig. 4). Similar arguments can be made regarding the complementation of Cdc4p-F12L and Cdc4p-G82D, with amino acid substitutions in their N-terminal and C-terminal domains, respectively. In the case of Cdc4p-F12L and Cdc4p-R33K, with mutations in the same domain, a wild-type complex with Myo2p may arise if one amino acid substitution disrupts the binding in the conformation required of an ELC but not an RLC, and vice versa for the second substitution. This situation is more akin to the case in which interallelic complementation arises when a protein has two independent functions, namely that of an ELC and RLC.
Functional Considerations--
Location- and
time-dependent assembly and activity of a contractile ring
are primary features of cytokinesis (1, 2, 12). The function of Cdc4p
as a light chain has been largely inferred by analogy to known
functions of myosin light chains in muscles. The muscle analogy may not
be entirely appropriate. The requirements in muscle cells for variable
speed and force development are unlikely features of cytokinesis.
Conversely, the contractile ring is a transient structure, assembled
and disassembled in minutes. We suggest that Cdc4p has the potential to
interact with Myo2p in the equivalent mode of both the ELC and RLC.
Does it follow from the muscle analogy that as an RLC, metal binding or
phosphorylation of Cdc4p controls the activity of the contractile ring?
There is currently no evidence for this. As shown in this study, Cdc4p
does not bind calcium. Furthermore, fission yeast expressing mutated
versions of Cdc4p that cannot be phosphorylated at serines 2 or 6 (Fig.
1) grow and divide normally (40). Could Cdc4p be involved in regulating
the assembly or disassembly of the contractile ring? Detection of Cdc4p
in improperly formed medial rings in Myo2p-deficient cells (13), as
well as potential interactions with Rng2p, suggest roles beyond that of
simply an ELC. Finally, what are the functional consequences of the
recently detected partnerships of Cdc4p with a putative PI 4-kinase and a Vps27p-like protein (49). The structure of Cdc4p and the proposed models for interallelic complementation provide several clear and
testable predictions for addressing these questions and thereby delineating the role of this protein in the assembly and function of
the contractile ring in fission yeast.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Paul Nurse for S. pombe strains cdc4-8 and cdc4-31 and Dr. Mohan Balasubramanian for
S. pombe strains cdc4A1, cdc4
A2, cdc4
A11, and
cdc4
C2. We also thank Les Hicks and Dr. Cyril Kay for running
equilibrium ultracentrifugation experiments on Cdc4p, Dr. Brian Sykes
for cTnC coordinates prior to publication and Dr. Leo Spyracopoulos for comments.
![]() |
FOOTNOTES |
---|
* This work was supported by grants from the Leukemia Research Fund (to C. M. S.), the Medical Research Council of Canada (to S. M. H.), the National Cancer Institute of Canada, and the Protein Engineering Network Centers of Excellence (to L. P. M.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1GGW) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
§ Present address: Protein Engineering Network Centres of Excellence, 713 Heritage Medical Research Centre, University of Alberta, Edmonton, Alberta T6G 2S2, Canada.
To whom correspondence may be addressed. Tel.: 306-975-5242;
Fax: 306-975-4839; E-mail: hemmings@cbrpbi.pbi.nrc.ca.
§§ An Alexander von Humbolt Fellow and a Canadian Institutes of Health Research Scientist. To whom correspondence may be addressed. Tel.: 604-822-3341; Fax: 604-822-5227; E-mail: mcintosh@otter.biochem.ubc.ca.
Published, JBC Papers in Press, November 21, 2000, DOI 10.1074/jbc.M008716200
2 M. K. Balasubramanian, personal communication.
3 The coordinates for the ensemble of structures, along with experimental restraints and NMR chemical shifts, have been submitted to the Protein Data Bank and the BioMagResBank.
4 L. F. Saltibus, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: ELC, essential light chain; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhancement spectroscopy; HSQC, heteronuclear single quantum correlation; RLC, regulatory light chain; 4-kinase, phosphatidylinositol 4-kinase; cTnC cardiac troponin C, r.m.s., root mean square; mutations are designated by the single letter codes for the wild-type and mutant sequences, respectively, separated by the residue number.
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