From the Centro de Investigaciones
Biológicas, Consejo Superior de Investigaciones
Científicas, C/Velázquez, 144, 28006 Madrid and the
¶ Centro Nacional de Biotecnología, Consejo Superior de
Investigaciones Científicas-Campus de Cantoblanco,
28049 Madrid, Spain
Received for publication, December 4, 2000
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ABSTRACT |
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The effect of bound nucleotide on the
conformation of cell division protein FtsZ from Methanococcus
jannaschii has been investigated using molecular dynamics and
site-directed mutagenesis. The molecular dynamics indicate that the
FtsZ and tubulin are proteins essential for cell division.
Tubulin FtsZ together with tubulin form a distinct group of GTPases (3-6),
which form in turn structural polymers. Tubulin assembles into
microtubules, hollow cylinders composed of longitudinal protofilaments (7), whereas FtsZ polymerizes in vitro into
microtubule-related filaments (8, 9). A third GTPase, dynamin (10),
whose possible structural relationship with FtsZ and tubulin is
presently unknown, shares with FtsZ organelle division functions (11, 12) and is able to in vitro self-assemble into ring-shaped
oligomers and helical polymers (13).
The structures of FtsZ and tubulin have a common fold and are
remarkably similar (14, 15). FtsZ and tubulin have a limited structural
similarity with the small G-proteins of the ras type (16).
FtsZ, tubulin, and the ras proteins also share the function of molecular switches activated by GTP. The activated protein is able
to interact with a target protein (downstream signal transmission), the
interaction being heterophilic in the cases of ras, and
homophilic in the case of the structural GTPases.
The lifetime of the activated state of these proteins is regulated by
the interaction with GTPase-activating proteins
(GAPs)1 (17), which provide
an external residue to the active center that stabilizes the existing
catalytic machinery (18) (upstream signal transmission). As in the case
of the downstream signal transmission, in ras this upstream
interaction is heterophilic, since the GAP, ras, and the
target are different proteins. In the case of the structural GTPases
FtsZ and tubulin, the upstream interaction is also homophilic, since
the GAP is as well FtsZ or tubulin (16). So the GAP-activated protein,
activated protein-target interactions are identical, with the net
result being the formation of a homopolymer. That means the
activation/deactivation process of the structural GTPases is
conceptually identical but biochemically much simpler than in the case
of ras-like proteins. The protein to which the signal is
transmitted and the one that provides the additional residue that
activates the GTPase are identical, i.e. another molecule of
tubulin or FtsZ. Thus, the structural GTPases constitute an ideal
system for studying molecular signal transmission as identical protein
molecules transmit, receive, and interrupt the signal transmission.
Information about the nature of the structural changes that the binding
of an activator induces in a protein molecule, and how the activation
signal is transmitted, can be obtained by comparing the structures of
the initial and final stages of the activation transition.
Nevertheless, little can be deduced about the mechanism of molecular
activation from the presently available x-ray crystal structure of
GDP-bound FtsZ from Methanococcus jannaschii (14) and the
3.7-Å resolution electron crystallographic structure of A useful approach to study conformational transitions in proteins is
the use of computational methods. Knowing the structure of GDP-bound
FtsZ, it is possible to introduce a The Methanococcus genus of methanogenic archea consists of
five species, all of them hyperthermophiles (25). The stability of
proteins in these archea is seriously compromised by the high temperatures of their habitats, the folded state of the proteins being
favored by a higher concentration of chaperones, the accumulation of
certain solutes like 2,3-cyclic diphosphoglycerate and certain modifications of the sequence that produce a larger number of salt
bridges and increase the hydrophobicity of the core (mutations that
introduce holes in the hydrophobic core of hyperthermophile proteins
seriously compromise their stability (26)). FtsZ from M. jannaschii should be expected to be extremely stable at the relative low temperature (300 K) at which the simulations are performed, so that any movements detected when the In this paper the solvated GDP and GTP-bound molecular
dynamics-calculated structures are compared with the crystal structure of FtsZ from M. jannaschii (14). Structural changes found in zones potentially functional in activation for assembly are here described. A single tryptophan mutant at loop T3 has been designed according to molecular dynamics predictions, whose fluorescence properties monitor the GDP or GTP nucleotide bound to the protein.
Protein Purification and
Characterization--
MgCl2, EDTA, KCl, and Tris were from
Merck. GDP was from Sigma and GTP (lithium salt) from Roche Molecular
Biochemicals. Guanidine hydrochloride was from Calbiochem. Other
analytical grade chemicals were from Merck or Sigma, except as
otherwise indicated.
FtsZ from M. jannaschii (FTSZ1 or MJ0370) was expressed in
Escherichia coli BL21/C41 as described (14). This wild type
FtsZ has a single tryptophan at position 319. The T92W-W319Y mutant FtsZ was constructed using a modified version of the inverse-PCR site-directed mutagenesis protocol of Ref. 27. The first step toward
producing this double mutant of FtsZ was to make the W319Y FtsZ mutant.
Inverse PCR was performed using the Excite High Fidelity PCR System
(Roche Molecular Biochemicals), the plasmid pHis17-mjFtsZ-H (28) as
template, and the following 5'-phosphorylated primers: p319Tyr
(ATATATAATTGTAGCATTTGGGTCTAATCTTG), which results in changing Trp319 to Tyr; and p319NarI (GGCGCCACAATAGATGAGAACTTAG),
which introduces an adjacent silent restriction site marker,
NarI, that is unique to the plasmid. The PCR parameters were
1 cycle of 5 min at 95 °C; followed by 15 cycles of 15 s at
95 °C, 30 s at 55 °C, and 4 min at 68 °C, terminating
with 1 cycle of 20 min at 68 °C. The PCR product was purified with
the Concert Rapid PCR Purification System (Life Technologies, Inc.) and
polished with the Pwo DNA polymerase (Roche Molecular
Biochemicals) to remove any additional nucleotides added to the 3' end
of the PCR product by the Excite High Fidelity polymerase. The polished
PCR product was purified, ligated by T4 DNA Ligase (Roche Molecular
Biochemicals), and then digested with DpnI enzyme (Promega)
to reduce the level of parental plasmid. The ligated PCR product was
then transformed into E. coli DH5
The proteins were purified as described in Ref. 14 except that the
E. coli cells containing the protein were lysed by five 30-s
sonication cycles instead of by heat shock. For comparative purposes
the wild type protein was as well purified by a heat shock (14). Both
proteins were stored at
The relative affinity of the proteins for GDP and GTP was checked by
incubating 25 µM FtsZ (WT or T92W-W319Y) for 1 h at
4 °C in buffer A (50 mM Tris, 50 µM EDTA,
10 µM GDP, pH 8.0), 500 mM KCl plus 5, 10, 50, 100, or
200 µM GTP. The excess nucleotide was removed by a chromatography in
a 5-ml Hitrap desalting fast protein liquid chromatography column
equilibrated in 50 mM Tris, 50 µM EDTA, 500 mM KCl, pH 8.0, containing 10 µM [GTP + GDP], where the GDP/GTP ratio was the same as in the incubation
mixture. The nucleotide content of the mixture was characterized by
HPLC (30).
Laser desorption/ionization mass spectroscopy measurements were
performed on a BIFLEX time-of flight instrument (Bruker-Franzen Analytik, Bremen, Germany) operated in the positive mode. Samples were
analyzed in the linear mode, and typically 100 laser shots were summed
into a single mass spectrum. External calibration was performed using
bovine serum albumin (Mr 66,432.9) as standard. The molecular mass values obtained were 39,817 ± 80 for the wild type (theoretical 39,891.2), and 39,975 ± 80 (theoretical
39,953.2) for the T92W-W319Y double mutant.
The fluorescence emission spectra of the proteins in their GDP- and
GTP-bound state were measured at 25 °C employing a Shimadzu RF-540
spectrofluorimeter (excitation wavelength, 295 nm; 5-nm excitation and
emission slits)
The circular dichroism spectra of the proteins (10 µM
FtsZ) equilibrated in Buffer A, 500 mM KCl, plus 50 µM GDP or GTP were measured at 25 °C employing a JASCO
J720 dichrograph, using 0.1-cm cells. The contents in the secondary
structure were estimated by deconvolution of the CD spectra using the
convex constraint algorithm method (32).
Sedimentation equilibrium and velocity measurements were performed at
25 °C in an Optima XL-A (Beckman-Coulter) analytical Ultracentrifuge
as described (33). Whole-cell apparent average molecular weights of
FtsZ were obtained using the program EQASSOC (34). Sedimentation
coefficients were calculated by global fitting of multiple
sedimentation profiles with the program SVEDBERG (retrieved from the
RASMB server (35)).
Molecular Dynamics--
The starting structure of the GDP form
(1FSZ) (14) of FtsZ from M. jannaschii was obtained from the
Brookhaven Protein Data Bank (36). The starting coordinates for the
simulation of the conformation of FtsZ bound to GTP were those of the
GDP form with a Quality of the Molecular Dynamics Simulations: r.m.s. Deviations
and Energy--
Fig. 1 (A and B) shows the
r.m.s. deviation of the simulated solution structures of GDP-bound and
GTP-bound FtsZ from the crystal structure of the GDP-bound form of the
protein. In five of the six simulations, an equilibration time of 200 ps is enough to reach plateau values (GDP: D1 1.64 ± 0.08 Å, D3
1.87 ± 0.14 Å; GTP: T1 1.75 ± 0.10 Å, T2 1.52 ± 0.06 Å, T3 1.90 ± 0.31 Å). The apparently different behavior of
one GDP simulation (D2, which reaches a rapid equilibrium, like the
others, after 60 ps, but between 300 and 400 ps undergoes a relatively
large deviation; see Fig. 1A)
is simply due to rotation around the
Thr36-Lys37 bond, which changes the position of
the helix H0 without altering its structure (data not shown). The
r.m.s. deviation of this simulation (D2), calculated without taking
into account helix H0, stabilizes after 300 ps at a plateau value of
1.55 ± 0.07 Å, similarly to the other simulations.
The last 50 ps of each simulation were averaged to obtain the final
average structures. The three final average structures with each
nucleotide (GDP: D1s, D2s, D3s; GTP: T1s, T2s, T3s) were compared and
found to be very similar except for some differences in the position of
the H0 helix, and of the loop from Ile251 to
Ala265 (r.m.s. deviation values: D1s-D2s 1.54 Å, D1s-D3s
1.43 Å, D2s-D3s 1.53 Å, T1s-T2s 1.61 Å, T1s-T3s 1.53 Å, T2s-T3s
1.49 Å).
Fig. 2 (A and B)
show the evolution of the potential energy of the systems. The initial
potential energy of both forms is equal in all simulations ( Comparison of the Calculated Solution Structures with the Crystal
Structure of FtsZ--
Six 500-ps molecular dynamics simulations of
M. jannaschii FtsZ bound to GDP (D1, D2, and D3 simulations)
and GTP (T1, T2, and T3 simulations) were performed to study the
conformational effects of the nucleotide bound to the protein (see
"Experimental Procedures"). Fig. 3
shows the deviation of the position of the C
First of all, the N-terminal
The GDP forms show less differences with the crystallographic structure
than the GTP forms (as expected since the crystallographic structure is
GDP-bound), the observed differences are mainly focused in the area
between Gly88 and Gly99. Other differences can
also be observed in the positions of the loops from Pro191
to Ala207, Asp257 to Glu259, and at
residue Ala282 (Fig. 3A). Nevertheless
differences are smaller than those observed for the GTP-bound forms.
The GTP-bound forms show larger conformational differences with the
crystal structure in the zone between Gly88 and
Gly99, and in the loop from Pro191 to
Ala207. Additional differences with the crystal structure
are located in the zones from Met58 to Ala64
and Lys109 to Ser111 (Fig. 3B). Fig.
4 shows all these zones labeled onto the
crystal structure of FtsZ.
Location of Nucleotide-induced Structural Changes--
Since
solvent exposed loops may adopt different conformational states, the
nine pairs of GDP and GTP structures have been compared to discriminate
the more systematic deviations. Fig. 5
shows the deviations between the D1s and T1s, D2s and T2s, and D3s and
T3s structures as representative examples. The only differences among
D-T pairs that can be systematically observed (at least in six of the
nine cases) are reduced to the regions
Met58-Ala64 (six of nine cases),
Gly88-Gly99 (nine of nine cases), and
Lys109-Ser111 (eight of nine cases). Among
these, the Gly88-Gly99 loop shows considerably
larger deviations. The deviations of the
Pro191-Ala207 loop are not so consistently
observed (only four of nine cases).
The GDP and GTP simulated structures have similar flexibilities, except
at two points: the segment Gly96-Gly99 and
Asn201. This fact has been observed in all six simulations.
Interestingly, Gly99 is one of the more flexible residues
in the GDP simulation, becoming more rigid in the GTP simulations. On
the other hand, Asn201 has increased flexibility in the GTP
simulations. As an example, Fig. 6 shows
the difference r.m.s. fluctuations of the C
Fig. 7 shows an insight of the
Gly88-Gly99 (loop T3) zone of the T1s and D1s
structures, the crystal structure of FtsZ, and the electron
crystallography structures of Modeling Mutations at Loop T3--
To prove whether the loop T3 of
FtsZ constitutes a switch area, a fluorescent probe may be introduced
in the loop, which should have different fluorescent properties in the
GDP- and GTP-bound states. This was the case, for example, in the Y32W
mutant of Ha-Ras-p21 (22) employed to monitor the activation state of that protein. Such a mutant with a fluorescent residue in the putative
effector loop would be of great interest for studying the activation
mechanisms of the FtsZ structural GTPase since the active and inactive
states could be detected with a simple spectrofluorometric test.
The loop T3 is very rich in small residues: 5 glycines and 1 alanine.
Since these residues have special allowed areas in the Ramachandran
plot, substitution of them by a large group would cause structural
perturbations. The loop is also rich in basic residues, 2 lysines and 1 arginine in 11 residues. Since mutating a charged residue in a
presumably active area may result in loss of activity, only three
candidate residues were left: Leu91, Thr92, and
Leu95. These three residues showed large differences (3-4
Å) in position between the crystal structure and both in
silico solution structures (Fig. 3), and even larger (about 6 Å)
between the GDP- and GTP-calculated structures (Fig. 5). The mutation
of these residues into the natural fluorophore, tryptophan, was modeled
into the final structures of the GDP and GTP simulations using the
WHATIF software package (44). In the resulting mutated model structures
the same large conformational differences in the position of the
tryptophans were observed. The larger differences (which may imply
larger differences in fluorescent properties) correspond to
Trp92, which is more exposed to the solvent in the
GTP-bound form than in the GDP-bound models. In addition, this residue
is sufficiently far away from the Wild Type and T92W-W319Y FtsZ from M. jannaschii Have Similar
Nucleotide Binding Capacity, Association State, and Secondary
Structure--
The wild type protein (purified by breaking the cells
by sonication) contained 0.70 mol of nucleotide/mol of FtsZ, of which 19.5% was GTP and 80.5% GDP. When purified by heat shock (15), it had
a similar nucleotide composition (0.80 nucleotide per FtsZ, 21.5% GTP
and 78.5% GDP). The T92W-W319Y FtsZ mutant contained less nucleotide
(0.28 nucleotide per FtsZ, 39.6% GTP and 60.5% GDP). However, both
proteins retained full nucleotide binding activity. The nucleotide
content increased both in the wild type and T92W-W319Y FtsZ up to 1.01 per FtsZ (3% GTP, 97% GDP in both cases) after 1 h of incubation
at 4 °C with 1 mM GDP and further equilibration in
buffer A plus 500 mM KCl.
The relative affinity of these proteins for GDP and GTP was checked as
described (see "Experimental Procedures"). Both the wild type and
the mutant exchange GDP for GTP with a similar affinity ratio of 4.25:1
(KGTP-FtsZ:KGDP-FtsZ)
(the total nucleotide content remained 1.01 per FtsZ). This affinity
ratio is modified by 1 mM MgCl2, to 1.45:1
(KGTP-FtsZ:KGDP-FtsZ).
Since it is known that tubulin binds divalent cations (45-47),
M. jannaschii FtsZ requires divalent cations to form
filaments (48) and FtsZ from E. coli requires
Mg+2 to hydrolyze GTP but not to assemble into filaments
(49) the content of Mg+2 and Ca+2 of the wild
type protein (both purification procedures) and of the double mutant
were measured. No Mg+2 or Ca+2 was found. This
was expected since the last step of purification is a gel filtration in
a buffer containing 1 mM EDTA, and coincides with the
crystallographic structure of FtsZ in which no metallic cations were
observed. The residual total Mg+2 and Ca+2 was
found to be lower than 1 and 5 µM, respectively, which
implies free concentrations below 13.8 and 1.3 nM,
respectively, in the 50 µM EDTA containing experimental
buffer employed.
The oligomerization state of the wild type and mutant protein was
studied using analytical ultracentrifugation. The apparent weight
average molecular weight was measured by sedimentation equilibrium at
different ionic strength and protein concentrations (Table
I). At high ionic strength, 500 mM KCl, both proteins are close to monomeric state if the
protein concentration is kept low enough. At low ionic strength, the
mutant has a lower self-association. To check if the nucleotide bound
to the protein may affect its aggregation state, sedimentation velocity
experiments were done. The measured sedimentation coefficients of WT
and mutant proteins in buffer A, 500 mM KCl, are not
affected by the nucleotide bound to them (Table
II). The values at 2 µM
FtsZ correspond to globular particles with a relative frictional
coefficient ratio of 1.2 ± 0.1.
To check whether the double mutation introduces a change in the
secondary structure of the protein, circular dichroism (CD) spectroscopy was performed. Both spectra from M. jannaschii
wild type and the double mutant FtsZ were identical (data not shown), indicating that no major change in secondary structure is caused by
these mutations. These spectra (data not shown) are typical for
Nucleotide-induced Changes in the Tryptophan Fluorescence of
T92W-W319Y FtsZ Monomers--
The single tryptophan residue
(Trp319) of M. jannaschii FtsZ is far away from
the nucleotide binding area. Fig. 8 shows
the tryptophan fluorescence emission spectra of the wild type and
T92W-W319Y mutant of FtsZ (2 µM monomers) in their GDP-
and GTP-bound states. Trp319 has a much larger quantum
yield than Trp92 (3-4 times more fluorescence intensity),
but its fluorescence is not modified by the nucleotide bound.
As predicted, the tryptophan introduced at position 92 is very
sensitive to the nucleotide bound. The tryptophan in the GTP state has
30% more fluorescence intensity than with GDP and its emission maximum
at 348 nm, while in the GDP state the maximum is at 343 nm (Fig.
8B). The change in intensity is linearly dependent on the
nucleotide content of the protein (Fig. 8C). Since the change in the emission maximum is not too large, this indicates that
the difference in intensity is due to the different quantum yields of
the GDP and GTP states of the FtsZ monomers. The same intensity change
was measured at higher FtsZ concentrations (20 µM).
Addition of up to 5 mM MgCl2 to the solutions
of Fig. 8 produces no appreciable change in the fluorescence of the
tryptophan neither in the GDP-bound nor in the GTP-bound states,
indicating that the binding of the divalent cation (which affects
GTP/GDP affinity ratio) does not influence the conformation of loop T3.
The fluorescence emission maximum of Trp92 shows a large
shift to the red compared with Trp319 and typical values in
native proteins (50), indicating a large accessibility of the residue
to the solvent (in the GTP-bound state, this residue has an emission
maximum coincident with that of tryptophan in water). The mean solvent
accessibility of the side chain of Trp92, modeled into the
GTP simulated structures, is 25.3 ± 1.2 Å2, and
18.1 ± 1.1 Å2 when modeled into the GDP structures.
The mean accessibility of the Trp319 side chain of the wild
type protein is lower, 15.8 ± 0.6 Å2. The positions
of the different maxima of the emission spectra (348 nm for T92W-W319Y
GTP-bound, 343 nm for T92W-W319Y GDP-bound, 335 nm for wild type) are
qualitatively concordant with these solvent accessibilities.
Conformational Differences between the Solution Structures of GDP
and GTP-bound FtsZ Calculated from Its Crystal Structure with Molecular
Dynamics--
The main goal of this work was to investigate how the
presence or absence of the
The simulated structures equilibrate relatively rapidly (in around 200 ps) and deviate very little from the x-ray structure, indicating the
quality of the simulation. The GDP-bound form shows a slightly lower
r.m.s. deviation from the x-ray determined structure than the GTP one
as expected. The energy of the calculated structures decreases quite
rapidly, indicating the quality of the refinement of the x-ray
structure. The GTP-bound calculated structures show lower energies,
which suggest increased stability of this form.
The calculated structures consistently deviate from the x-ray structure
in the loop from Gly88 to Gly99. This zone
deviates up to 4 Å for some residues in the GDP-bound calculated
structures and up to 6 Å in the GTP-bound ones. This loop is quite
well defined in the crystallographic structure and shows the lowest
B-factors of the molecule, so that the large differences
calculated should indicate an influence of the nucleotide in the
conformation of the loop.
The structure of the loop in the calculated solvated GDP form is
slightly different from the crystal structure. The nucleotide bound to
the purified protein is mainly GDP (80%); nevertheless, a significant
percentage of the protein (20%) has GTP bound, which explains why a
weak electron density of the
The molecular dynamics results strongly suggest that the structure of
the zone between Gly88 and Gly99 depends on the
presence of the nucleotide
Loop T3 is apparently able to transmit the signal of the presence of
the Loop T3 as Switch Element of FtsZ and Tubulin--
To confirm the
molecular dynamics predictions, a mutant in which a fluorescent probe
is introduced at loop T3 of FtsZ was constructed. The single tryptophan
of the protein at position 319 has been replaced by a tyrosine, and a
new tryptophan has been introduced at position 92. The mutant behaves
as predicted, showing appreciable changes in fluorescent intensity and
in the position of the tryptophan emission maximum depending on the
bound nucleotide. Since from the relevant tryptophan quenchers that can
be found in proteins (disulfide bridges, protonated histidine, cysteine, tyrosine, and carbonyl carbons; Ref. 53) only the carbonyl
carbons of the peptide bonds are present in the environment of
Trp92, the change in fluorescence intensity should really
indicate a conformational change of the main chain of the loop. The
5-nm blue shift of the emission maximum is interpreted as an increase in exposure to the solvent of the Trp side chain, as observed in the
molecular dynamics model structures (see "Results").
Loop T3 is highly homologous between FtsZ and tubulin, although the
first part of the loop is seven residues longer in FtsZ (16). Since the
x-ray structure of a GTP-bound form of FtsZ is not presently available,
it is not possible to compare the structure of loop T3 in the GTP- and
GDP-bound states of FtsZ. Nevertheless, the GTP-bound and GDP-bound
calculated structures can be compared with the GDP-bound FtsZ x-ray
structure and with the
The comparison of the GDP and GTP calculated average structures (Fig.
7A) and the crystal structure of FtsZ (Fig. 7B)
shows that the position of the loop T3 in the GDP-bound form is even more displaced toward the outer part of the protein than in the crystal
structure, whereas the structure of the GTP-bound form is more
displaced toward the nucleotide in central part of the interface. This
points out that the forces produced by the presence of the
FtsZ monomers have been modeled into an electron microscopy density map
of FtsZ filaments (48), using as a starter model the structure of the
tubulin dimer (15). In this manner monomer features may be applied to
the polymer. Fig. 9 shows a model of FtsZ-FtsZ contact constructed as described (48), using the crystal structure of 1FSZ. The view is equivalent to a microtubule
protofilament seen from outside the microtubule. As can be seen, loop
T3 is located at one side of the putative contact interface between two
FtsZ molecules. Loop T3 has contacts with the beginning of helix H8 and
a collision with the end of loop T7 of the contacting FtsZ molecule as
described (48). Additional contacts can be observed with helix H0, but
as seen in this work the helix has a relatively large conformational
freedom, and it is probably in a different position in the filament, as
pointed out by the fact that helix H0 density does not fit in the
electron microscopy map and must be moved to fill the empty part of the
density, which lies nearby (48).
Fig. 9 (B-D) shows a detail of the loop T3-helix H8 contact
area in the same model of FtsZ-FtsZ association (Fig. 9A)
built with the GDP, GTP, and crystal structures. The contact area in T1s (Fig. 9C) is very similar to the one of the crystal
structure model (Fig. 9D) (minor collisions with helix H8
and loop T7 are observed). However, the conformation of loop T3 in the
D1s structure (Fig. 9B) should push helix H8 upward, thus
bending the filament; otherwise, large superpositions of both main
chains (represented by the red coloring) arise. It is thus
conceivable that loop T3 conformation, which can switch between the
characteristic GTP and GDP states, may modulate the bending of the FtsZ
filament, inhibiting assembly. Note that these representations are just models since an accurate simulation of the effect of the conformational changes in the interface would require accurate structures of the
contact surface, which are not available. In any case, there is
evidence that GTP favors the straight FtsZ filament conformation and
GDP the curved conformation, so that GTP hydrolysis might be used to
generate force for the constriction of the FtsZ ring during cell
division (54).
A comparison between the structure of loop T3 in FtsZ, -phosphate of GTP induces a conformational perturbation in loop T3
(Gly88-Gly99 segment), in a position
structurally equivalent to switch II of Ha-ras-p21. In the
simulated GTP-bound state, loop T3 is pulled by the
-phosphate into
a more compact conformation than with GDP, related to that observed in
the homologous proteins
- and
-tubulin. The existence of a
nucleotide-induced structural change in loop T3 has been confirmed by
mutating Thr92 into Trp (T92W-W319Y FtsZ). This tryptophan
(12 Å away from
-phosphate) shows large differences in fluorescence
emission, depending on which nucleotide is bound to FtsZ monomers. Loop
T3 is located at a side of the contact interface between two FtsZ
monomers in the current model of FtsZ filament. Such a structural
change may bend the GDP filament upon hydrolysis by pushing against
helix H8 of next monomer, thus, generating force on the membrane during cell division. A related curvature mechanism may operate in
tubulin activation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-dimers self-assemble into eukaryotic microtubules (1), whereas FtsZ is a main component of the prokaryotic septation ring (2).
The functions of these proteins make them obvious targets for antitumor
drugs (tubulin) or for a possible new generation of antibiotics (FtsZ).
-tubulin
in zinc-induced polymers (15).
-phosphate into the molecule and
study the perturbations induced in the protein structure. The
computational methods employed are based on molecular dynamics
(19-21), in which simulations of the solution structures with GDP and
GTP bound on the nucleotide site are compared. In principle, a long
enough molecular dynamics simulation should be able to find the
structures of the inactive GDP-bound form (which may diverge from the
starting crystal structure), and of the active GTP-bound form of FtsZ.
Unfortunately, the calculations may tend to stay in local minima and
the equilibration time needed might be much longer than the maximum
simulation time presently affordable (nanosecond). Nevertheless, it is
still possible to obtain useful information about the system by
studying the perturbations induced in the structure by the presence of
the
-phosphate. These procedures were successfully employed, in
combination with targeted molecular dynamic techniques (19), to predict
the hinges of the conformational transition between the active and the
inactive state of Ha-ras-p21 (20, 21). These predictions
have been experimentally confirmed (22-24).
-phosphate group
is introduced into the structure should be probably more related with
the activation state of the protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
cells and the desired
transformants selectively grown on LB plates containing 50 µg/ml
ampicillin and 50 µg/ml chloramphenicol. Plasmid DNA was purified
from individual transformants using the Concert Rapid Plasmid
Purification system (Life Technologies, Inc.), and the mutant plasmid,
pAVK1(W319Y) identified by digestion with NarI, followed by
the sequencing of the gene for FtsZ with sequencing primers mf3
(ACCATATCTGAATCTTG), mj1r (CGGCATATTTGGAAC), and p319Tyr. The double
mutant of FtsZ was then obtained in an identical manner, except the PCR
template was pAVK1(W319Y) and the primers were pHindT92W
(TAAAAAGCTTTGGAGAGGTCTTGGAGC), which results in changing
Thr92 to Trp and introduces the adjacent silent restriction
marker, HindIII (of which there is one other site in the
plasmid), and pantiHind (CCAATTAATATTTTTTTATCAGC). The double
mutant plasmid, pAVK2 (T92W-W319Y) was identified by HindIII
digestion, sequenced as before, and transformed into E. coli
BL21:pLys for overexpression by induction with 1 mM
isopropyl-1-thio-
-D-galactopyranoside.
70 °C and equilibrated in the desired
buffer prior to use. Protein purities were checked by
SDS-polyacrylamide gel electrophoresis (29) and were found to be >98%
for all three purified proteins. Protein concentrations were measured
by taking their UV spectra in 6 M guanidine hydrochloride, after subtracting the contribution of nucleotide, which was measured and characterized by HPLC as described (30). The extinction coefficients employed (280 nm) were 8100 M
1 cm
1
for guanosine nucleotides,2
and 1280 M
1
cm
1 for tyrosine and 5690 M
1 cm
1
for tryptophan (31). The total Mg+2 and Ca+2
concentrations were measured by atomic absorption spectrometry with a
PerkinElmer Life Sciences model 2380 spectrometer.
-phosphate added to the GDP (the coordinates of the
-phosphate were obtained by overimposing the GDP bound to FtsZ with
the GTP bound to the
-tubulin subunit (1TUB); Ref. 15). Since no
Mg+2 was found to be bound to the purified protein as
described by Löwe and Amos (14), and no Mg+2
coordinates were available in the
-tubulin heterodimer (15), the
uncertainty in the position of possible Mg+2 cation bound
to the protein was too high to model it in the nucleotide site. The
GROMOS 96 software package (37) was obtained from BIOMOS b.v.
(Groningen, The Netherlands). The Protein Data Bank format of the
structure files was transformed into GROMOS 96 format using the
procs2 routine of the software package, then the polar hydrogen coordinates were generated using progch, and the
resulting structure was included into a 87-Å-wide truncated octahedral
water box of single point charge water (38) where a minimum distance of
8 Å was kept between the protein and the border of the box. The energy
of the resulting structure was minimized for 500 steps using a steepest
descent algorithm (39), and counterions (Cl
and
Na+) were added to neutralize the charges of the system.
The systems simulated with GDP and GTP bound contained 31,162 and
31,158 atoms, respectively. The system was then energy-minimized for
another 500 steps. The minimized structures were found not to diverge from the Protein Data Bank structure (r.m.s. deviation 0.01 and 0.02 Å for the GDP- and GTP-bound minimized structures, respectively). The velocities of the atoms were then randomly assigned to a Maxwellian velocity distribution at 300 K and three different free molecular dynamics simulation of each system (D1, D2, and D3 for the GDP-bound structure and T1, T2, and T3 for the GTP-bound structure), with different starting Maxwellian velocity distributions assigned, were
performed for 500 ps using a constant pressure of 1 atm and a constant
temperature of 300 K. The temperatures of the protein and the solvent
were separately coupled to a water bath (40) using a coupling constant
of 0.1 ps. The pressure was kept constant by coupling to an external
pressure bath (40) with a coupling constant of 0.5 ps. The conditions
of the MD simulation were the following: the time step employed was 2 fs, the integration of the equations of motion was done using the
leapfrog algorithm included in the GROMOS 96 package, the bond lengths
were constrained to equilibrium values using the SHAKE routine (41,
42), and a cutoff of 8 Å was used for nonbonded interactions and 14 Å for electrostatic interactions. For analysis the coordinates and
velocities were saved every 0.5 ps. For the calculation of the r.m.s.
deviation, the structures were fitted using a least-squares fit of the
C
atoms. The calculations were performed using the parallelized version of promd in a Challenge 10000 Silicon Graphics work-
station equipped with two MIPS R10000 processors. Each 500-ps
simulation required 80 days (160 days of CPU time). The data were
analyzed using SIMLYS version 3 (43).
View larger version (21K):
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Fig. 1.
r.m.s. deviation of the C
carbons of the calculated solution GDP-bound (A) and
GTP-bound (B) FtsZ structures from the x-ray determined
structure during the molecular dynamics simulations. Solid
line, D1 and T1 simulations; dashed
line, D2 and T2 simulations; dotted
line, D3 and T3 simulations.
395 MJ
mol
1) and drops very rapidly (1 ps) to values
of approximately
444 MJ mol
1, then an
equilibration time of 100 ps is required to reach a slightly lower
plateau for the GTP simulations than for the GDP simulations (T1
450.7 ± 0.5 MJ mol
1, T2
450.8 ± 0.5 MJ mol
1, T3 -450.5 ± 0.8 MJ
mol
1, D1 -449.6 ± 0.6 MJ
mol
1, D2 -449.9 ± 0.5 MJ
mol
1, D3
449.7 ± 0.5 MJ
mol
1).
View larger version (39K):
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Fig. 2.
Potential energies of GDP-bound
(A) and GTP-bound (B) FtsZ during the molecular
dynamics simulations. Solid line, D1 and T1
simulations; dashed line, D2 and T2 simulations;
dotted line, D3 and T3 simulations.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
atoms of each residue
of the GDP-bound final average solution structure of the protein after
the D1 simulation (D1s structure) and GTP-bound final average solution
structure of the protein after the T1 simulation (T1s structure) with
respect to the crystallographic structure of FtsZ (Xs). The deviations
observed in the other GDP (D2s and D3s) and GTP average structures (T2s
and T3s) from the crystal structure are similar to those shown (Fig.
3C shows the deviation between two GTP simulations (T1-T3)
as a representative control). These changes can be qualitatively
described as follows.
View larger version (30K):
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Fig. 3.
Deviation between the positions of the
C atoms of the averaged calculated solution
structures of FtsZ bound to GDP (D1s structure) (A) and
bound to GTP (T1s structure) (B) with respect to the
crystal structure; deviation between two GTP-bound calculated
structures T1s and T3s (C) is shown as comparison. The
solid lines represent the r.m.s. deviation of the
calculated structures from the x-ray determined structure, the
dotted lines represent the r.m.s. deviation plus
a S.D., and the vertical dashed lines
mark the H0 helix area, which has a large deviation due to the
conformational freedom of Lys39-Ile40
bond.
-helix H0, spanning from
Ser23 (the first amino acid present in the crystallographic
structure) to Lys39, shows large positional differences
among all structures (D1s, D2s, D3s, T1s, T2s, T3s, and Xs) due to the
rotational freedom of the Lys39-Ile40 bond.
Nevertheless the structure of the
-helix is completely stable.
View larger version (59K):
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Fig. 4.
Ribbon representation of the x-ray structure
of FtsZ on which the relevant structural elements described in the text
have been marked (drawing generated with the program MOLSCRIPT; Ref.
58).
View larger version (31K):
[in a new window]
Fig. 5.
Deviation between the positions of the
C atoms between three different pairs of
averaged solution structures of FtsZ bound to GDP and GTP. Figure
shows deviation between structures D1s and T1s (A),
deviation between structures D2s and T2s (B), and deviation
between D3s and T3s structures (C). The solid
lines represent the r.m.s. deviation between the calculated
GDP-bound and GTP-bound structures, the dotted
lines represent the r.m.s. deviation plus a S.D., and the
dashed lines mark the H0 helix area, which has a
large deviation due to the conformational freedom of
Lys39-Ile40 bond.
carbon atoms of the GDP-
and GTP-bound structures during the last 200 ps of the D1 and T1
simulations (as the fluctuations of the molecule equilibrate after 150 ps of simulation, the last 200 ps were judged to provide a stable
structure).
View larger version (21K):
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Fig. 6.
Difference in the r.m.s. fluctuations of the
C atoms of the calculated FtsZ GDP- and
GTP-bound forms during the last 200 ps of the D1 and T1
simulations. Positive values indicate increased flexibility in the
D1 simulation, negative values increased flexibility in the T1
simulation.
- and
-tubulin. The comparison shows this loop being pulled into a more compact conformation in the
presence of the GTP
-phosphate. A hydrogen bond is formed in the T
model between the
-phosphate of the GTP and the backbone N-H of
Gly99 of FtsZ (the N-O distances are 2.7 Å in T1s, 3.2 Å in T2s, and 3.1 Å in T3s). The
-phosphate of the GDP is too far
away to interact with this NH group (the N-O distances are 4.5 Å D1s,
4.2 Å D2s, 5.5 Å D3s). The loop in the crystal structure (GDP) is, as
expected, more close to the GDP model structure than to the GTP model
structure.
View larger version (16K):
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Fig. 7.
Detail of the conformation of loop T3.
Superposition of loop T3 of the FtsZ GTP-bound T1s structure
(yellow), with the one of the GDP-bound D1s structure
(green) (A), loop T3 in the x-ray structure of
FtsZ (red) (B), superposition of loop T3 of the
-tubulin monomer (GTP-bound, yellow), with the one of the
-tubulin monomer (GDP-bound, green) (C).
-phosphate (11.5 ± 2.1 Å in
the GTP conformations) and
-phosphate (18.5 ± 3.1 Å in the
GDP conformations), so that potential changes in its fluorescent
properties can be assigned to conformational changes in the tryptophan
environment. M. jannaschii FtsZ contains one tryptophan at
position 319, which must be removed to have a single tryptophan mutant
whose spectral changes are easier to interpret. The double mutant
T92W-W319Y was constructed, expressed, and purified as described under
"Experimental Procedures."
Average molecular mass of M. jannaschii FtsZ (sedimentation
equilibrium)
Sedimentation coefficients of M. jannaschii FtsZ (sedimentation
velocity)
-proteins and are actually similar to those measured for E. coli FtsZ (33) and tubulin (6). The secondary structure content
estimated using the convex constraint algorithm method (32 ± 4%
-helix, 19 ± 9%
-sheet, 45 ± 8% other) is
almost identical to that of tubulin (6).
View larger version (13K):
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Fig. 8.
A and B, fluorescence
emission spectra ( exc = 295 nm) of 2 µM WT-FtsZ (A) from M. jannaschii
in buffer A 500 mM KCl, plus 50 µM GDP
(solid line), or plus 50 µM GTP (dashed
line) and 2 µM T92W-W319Y-FtsZ (B) in
buffer A 500 mM KCl, plus 50 µM GDP
(solid line) or plus 50 µM GTP
(dashed line). C, fluorescence
intensity
exc = 295 nm,
ems = 348 nm) of 2 µM
T92W-W319Y-FtsZ in buffer A (500 mM KCl, plus 50 µM guanine nucleotide). GDP and GTP mixed in different
proportions shown in the upper x
axis). The percentage of GTP in the total nucleotide content
of FtsZ is represented in the lower x
axis).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-phosphate of the nucleotide bound to its site on FtsZ affects the protein conformation. To do this, six 500-ps
free molecular dynamics simulations were performed, with either GDP or
GTP bound at the nucleotide site, employing the coordinates of the
x-ray determined structure of the GDP-bound protein.
-phosphate could be observed in fresh
crystals (14). In the crystal structure, loop T3 is packed with
contacts with other two molecules; contacts include the areas from
Leu29 to Ala38 in the H0 helix and other
residues in the vicinity of this helix (Asp121,
Asp123, Lys148, Leu153,
Asp235, Lys240) of one molecule, and a part of
the loop T3 and the nucleotide binding cup
Gly166-Arg169 of other molecule. It might be
possible that this loop is fixed in one of its possible conformations
by these crystal restraints, since neither heterogeneity and nor large
conformational changes between fresh and old crystals were observed
(14). Nevertheless, it must be pointed out that the conformation of the
loop in the GDP-bound state is more close to the crystallographic than
the GTP-bound one, and the conformation of the loop in the solvated calculated structures is very trustable since three independent simulations gave the same result.
-phosphate. Several residues in this loop
deviate about 6 Å between the calculated GDP and GTP structures. This
deviation is probably due to the hydrogen bond that is formed between
the
-phosphate of GTP bound and the N-H group of residue
Gly99 (Fig. 7A), which fixes this residue
therefore decreasing its flexibility (Fig. 6). This pulls on the T3
loop and stabilizes a more closed conformation similar to the one
observed in the tubulin monomers which are fixed in the active state.
The
-phosphate of the GDP is too far away to form this H-bond (Fig.
7, A and B). Although there is not an exact
equivalent of loop T3 in Ha-ras-p21, its position in the
FtsZ molecule is equivalent to that occupied by the switch II region of
this GTPase (which has a fold partially resembling the GTP-binding
domain of FtsZ and tubulin although its topology is different).
-phosphate to a small area
Lys109-Ser111, which forms a hinge between the
two segments of helix H3 (structurally equivalent to helix
2 of
Ha-ras-p21). Both segments of the helix form a 110° angle
in the crystal FtsZ structure, while in the electron crystallography
tubulin structure they form an approximate angle of 150° (both
monomers). In the GTP-bound modeled structures T1s, T2s, and T3s, this
hinge opens and the helical segments form an angle of 125 ± 5°,
while in the GDP-bound simulations the hinge closes to render a
100 ± 5° angle between both segments. Changes in length and
orientation in the structurally equivalent (although much shorter)
2
helix of Ha-ras-p21 upon GTP binding can be observed between
both crystal structures (the helix is longer and is more closely packed
with the
-sheet in the GTP-bound structure) (51, 52). This may
suggest a related intramolecular signal transmission mechanism. There
is a distant FtsZ zone, from Met58 to Ala64,
that also seems to be sensitive to the presence of the
-phosphate of
the nucleotide. Note that other zones (including possible domain movement), which have not been detected by the molecular dynamics analysis, may change as well in response to the presence of the
-phosphate.
- and
-tubulin structures (15). The latter
should correspond to an active state, since
-tubulin is bound to GTP
and
-tubulin is fixed in a Zn2+-induced polymer grown
from GTP-bound tubulin and stabilized with taxol.
-phosphate should change the conformation of the loop toward this
position in the active form of the protein, which is fully supported by
the fluorescence results with the T92W FtsZ mutant.
View larger version (77K):
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Fig. 9.
A, model of FtsZ association constructed
as described by Löwe and Amos (48), using the crystal structure
1FSZ as monomer. B, detail of the area of collision between
loop T3, helix H8, and loop T7 in a model of FtsZ association
constructed in the same way using the GDP-bound averaged calculated D1s
structure as monomer. C and D, detail of the same
area in the model constructed using the GTP-bound averaged calculated
T1s structure as monomer (C), and using the crystal
structure 1FSZ as monomer (D). Note that the area of
collision around the nucleotide binding is represented in a slightly
different perspective for better visualization purposes. The residues
are represented in colors indicating the degree of the collision.
Gray means no collision, blue means structural
collision between the side chain of the residues, green
means minor structural collision between the backbones (distance
between the atoms larger than 1 Å less than the sum of Van der Waals
radii), yellow means large structural collision between the
backbones (distance between the atoms between 1 and 2 Å less than the
sum of Van der Waals radii), and red means superposition of
the backbones (distance between the smaller than 2 Å less than the sum
of Van der Waals radii).
- and
-tubulin (Fig. 7) shows different conformations of the loop. In the
GDP-bound FtsZ in inactive conformation, loop T3 is displaced toward
the outer part of the nucleotide binding domain, while the conformation
of the loop in the active conformation of tubulin is displaced toward
the inner part of the molecule and it is more compact. Nevertheless,
this comparison has to be taken with care due to the gap present in the
tubulin sequences. Loop T3 of tubulin may participate both in the
longitudinal contact interface between tubulin dimers in the
protofilament as well as in the contact between adjacent protofilaments
in the microtubule (16, 55). Therefore, its conformation may easily
control the assembly of the tubulin molecule. If the position of loop
T3 in GDP-tubulin were similar to the one predicted by the simulation
for FtsZ, it might bend the protofilament in a direction perpendicular
to its axis. Structures resulting from bending of protofilaments have
been actually observed in the small angle x-ray scattering structure of
tubulin double rings assembled from GDP-tubulin (56) and in the 4-Å
crystal structure of the complex of two GDP-tubulin dimers with a
stathmin-like domain (57), and proposed to reflect the structural
change between the active (straight) and inactive (curved)
conformations of tubulin. Nevertheless, it must be pointed out that the
bending direction observed is different in both cases. In GDP-induced
tubulin double rings, this bending appears tangent to the microtubule
surface, as is predicted to be induced by the movement of loop T3,
whereas, in the stathmin complex, the tubulin curvature appears in an
oblique with respect to the microtubule surface.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Dr. Jan Löwe for kindly providing the plasmid containing FtsZ from M. jannaschii, Dr. German Rivas (Centro de Investigaciones Biológicas (CIB)) for support and useful discussions Dr. Alicia Prieto (CIB) for the mass spectroscopy, Dr. Carlos Alfonso (CIB) and S. Zorrilla for analytical ultracentrifugation experiments, Pilar Palacios (Centro Nacional de Biotecnología) for technical assistance, and the CIB biocomputing service for computing time.
![]() |
FOOTNOTES |
---|
* This work was supported in part by Comisión Interministerial de Ciencia y Tecnología (Spain) Grants BIO99-0859-C03-02-03 and BIO97-1246 and by Programa de Grupos Estratégicos de la Comunidad Autónoma de Madrid.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.
§ Recipient of a contract from Programa de Incorporación de Doctores a Grupos de Investigación en España. To whom correspondence should be addressed. Tel.: 34-915611800 (ext. 4380); Fax: 34-915627518; E-mail: fer@akilonia.cib.csic.es.
¶ Recipient of a fellowship from Programa de Estancia de Científicos y Tecnólogos Extranjeros en España.
Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010920200
2 J. F. Díaz and J. M. Andreu, unpublished data.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are: GAP, GTPase-activating protein; PCR, polymerase chain reaction; HPLC, high performance liquid chromatography; WT, wild type; r.m.s., root mean square.
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REFERENCES |
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---|
1. | Lee, J. C., and Timasheff, S. N. (1975) Biochemistry 14, 5183-5187[Medline] [Order article via Infotrieve] |
2. | Bi, E., and Lutkenhaus, J. (1991) Nature 354, 161-164[CrossRef][Medline] [Order article via Infotrieve] |
3. | De Boer, P., Crossley, R., and Rothfield, L. (1992) Nature 359, 254-256[CrossRef][Medline] [Order article via Infotrieve] |
4. | RayChaudhuri, D., and Park, J. T. (1992) Nature 359, 251-254[CrossRef][Medline] [Order article via Infotrieve] |
5. | Sage, C. R., Dougherty, C. A., Davis, A. S., Burns, R. G., Wilson, L., and Farrel, K. W. (1995) Biochemistry 34, 7409-7419[Medline] [Order article via Infotrieve] |
6. | De Pereda, J. M., Leynadier, D., Evangelio, J. A., Chacón, P., and Andreu, J. M. (1996) Biochemistry 35, 14203-14215[CrossRef][Medline] [Order article via Infotrieve] |
7. | Amos, L., and Klug, A. (1974) J. Cell Sci. 14, 523-549[Medline] [Order article via Infotrieve] |
8. | Mukherjee, A., and Lutkenhaus, J. (1994) J. Bacteriol. 176, 2754-2758[Abstract] |
9. |
Erickson, H. P.,
Taylor, D. W.,
Taylor, K. A.,
and Bramhill, D.
(1996)
Proc. Natl. Acad. Sci. U. S. A.
93,
519-523 |
10. | McNiven, M. A., Cao, I., Pitts, K. R., and Yoon, I. (2000) Trends Biochem. Sci. 25, 115-120[CrossRef][Medline] [Order article via Infotrieve] |
11. |
Erickson, H. P.
(2000)
J. Cell Biol.
148,
1103-1105 |
12. | Margolin, W. (2000) Curr. Biol. 10, 328-330[CrossRef] |
13. | Hinshaw, J. E., and Schmid, S. L. (1995) Nature 374, 190-192[CrossRef][Medline] [Order article via Infotrieve] |
14. | Löwe, J., and Amos, L. A. (1998) Nature 391, 203-206[CrossRef][Medline] [Order article via Infotrieve] |
15. | Nogales, E., Wolf, S. G., and Downing, K. (1998) Nature 391, 199-203[CrossRef][Medline] [Order article via Infotrieve] |
16. | Nogales, E., Downing, K. H., Amos, L., and Löwe, J. (1998) Nat. Struct. Biol. 5, 451-458[Medline] [Order article via Infotrieve] |
17. | Adari, H., Lowy, D. R., Willumsen, B. M., Der, C. J., and McCormick, F. (1988) Science 240, 518-521[Medline] [Order article via Infotrieve] |
18. | Scheffzek, K., Reza-Ahmadian, M., and Wittinghoffer, A. (1998) Trends Biochem. Sci. 23, 257-262[CrossRef][Medline] [Order article via Infotrieve] |
19. | Schlitter, J., Engels, M., Krüger, P., Jacoby, E., and Wolmer, A. (1993) Mol. Sim. 10, 291-309 |
20. | Díaz, J. F., Wroblowski, B., and Engelborghs, Y. (1995) Biochemistry 34, 12038-12047[Medline] [Order article via Infotrieve] |
21. | Díaz, J. F., Wroblowski, B., Schlitter, J., and Engelborghs, Y. (1997) Proteins Struct. Funct. Genet. 28, 434-451[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Díaz, J. F.,
Sillen, A.,
and Engelborghs, Y.
(1997)
J. Biol. Chem.
272,
23138-23143 |
23. | Kuppens, S., Díaz, J. F., and Engelborghs, Y. (1999) Protein Sci. 8, 1860-1866[Abstract] |
24. | Díaz, J. F., Escalona, M. M., Kuppens, S., and Engelborghs, Y. (2000) Protein Sci 9, 361-368[Abstract] |
25. | Madigan, M. T., Martinko, J. M., and Parker, J. (1997) Brock Biology of the Microorganisms , pp. 741-768, Prentice Hall, Inc., Hertfordshire, U. K. |
26. | Fusi, P., Goosens, K., Consonni, R., Grisa, M., Puricelli, P., Vecchio, G., Vanoni, M., Zetta, Z., Heremans, K., and Tortora, P. (1997) Proteins Struct. Funct. Genet 25, 381-390[CrossRef] |
27. | Weiner, M. P., Costa, G. L., Schoettlin, W., Cline, J., Mathur, E., and Bauer, J. C. (1994) Gene (Amst.) 151, 119-123[CrossRef][Medline] [Order article via Infotrieve] |
28. | Löwe, J. (1998) J. Struct. Biol. 124, 235-243[CrossRef][Medline] [Order article via Infotrieve] |
29. | Laemmli, U. K. (1970) Nature 227, 680-685[Medline] [Order article via Infotrieve] |
30. | Díaz, J. F., and Andreu, J. M. (1993) Biochemistry 22, 2747-2755 |
31. | Pace, C. N., and Schmid, F. X. (1997) in Protein Structure: A Practical Approach (Creighton, T., ed), 2nd Ed. , pp. 253-259, Oxford University Press, Oxford |
32. | Perczel, A., Park, K., and Fasman, G. D. (1992) Anal. Biochem. 203, 83-93[Medline] [Order article via Infotrieve] |
33. |
Rivas, G.,
López, A.,
Mingorance, J.,
Ferrándiz, M. J.,
Zorrilla, S.,
Minton, A. P.,
Vicente, M.,
and Andreu, J. M.
(2000)
J. Biol. Chem.
275,
11740-11749 |
34. | Minton, A. (1994) in Modern Analytical Ultracentrifugation (Schuster, T. , and Laue, T., eds) , pp. 81-93, Birkhauser Boston, Inc., Cambridge, MA |
35. | Philo, J. S. (1997) Biophys. J. 72, 435-444[Abstract] |
36. |
Berman, H. M.,
Westbrook, J.,
Feng, Z.,
Gilliland, G.,
Bhath, T. N.,
Weissig, H.,
Shindyalov, I. N.,
and Bourne, P. E.
(2000)
Nucleic Acids Res.
28,
235-242 |
37. | Van Gunsteren, W. F., Billeter, S. R., Eising, A. A., Hünenberger, P. H., Krüger, P., Mark, A. E., Scott, W. R. P., and Tironi, I. G. (1997) Biomolecular Simulation: The GROMOS 96 Manual and User Guide , BIOMOS b.v., Zürich |
38. | Berendsen, H. J. C., Postma, J. P. M., Van Gunsteren, W. F., and Hermans, J. (1981) in Interaction Models for Water in Relation to Protein Hydration Intramolecular Forces (Pullman, B., ed) , pp. 331-342, Reidel, Dordrecht, The Netherlands |
39. | Levitt, M., and Lifson, S. (1969) J. Mol. Biol. 46, 269-279[Medline] [Order article via Infotrieve] |
40. | Berendsen, H. J. C., Postma, J. P. M., Van Gunsteren, W. F., Dinola, A., and Haak, J. R. (1984) J. Chem. Phys. 81, 3684-3690[CrossRef] |
41. | Ryckaert, J. P., Ciccotti, G., and Berendsen, H. J. C. (1977) J. Comput. Phys. 23, 327-341 |
42. | Van Gunsteren, W. F., and Berendsen, H. J. C. (1977) Mol. Phys. 34, 1311-1327 |
43. | Krüger, P., Lüke, M., and Szameit, A. (1991) Comput. Phys. Commun. 62, 371-380[CrossRef] |
44. | Vriend, G. (1990) J. Mol. Graphics 8, 52-56[CrossRef][Medline] [Order article via Infotrieve] |
45. | Lee, J. C., and Timasheff, S. N. (1977) Biochemistry 16, 1754-1764[Medline] [Order article via Infotrieve] |
46. |
Correia, J. J.,
Baty, L. T.,
and Williams, R. C., Jr.
(1987)
J. Biol. Chem.
262,
17278-17284 |
47. |
Menéndez, M.,
Rivas, G.,
Díaz, J. F.,
and Andreu, J. M.
(1998)
J. Biol. Chem.
273,
167-176 |
48. |
Löwe, J.,
and Amos, L. A.
(1999)
EMBO J.
18,
2364-2371 |
49. |
Mukherjee, A.,
and Lutkenhaus, J.
(1999)
J. Bacteriol.
181,
823-832 |
50. | Lakowicz, J. R. (1984) Principles of Fluorescence Spectroscopy , pp. 341-381, Plenum Press, New York |
51. | Tong, L. A., de Vos, A. M., Milburn, M. V., and Kim, S. H. (1991) J. Mol. Biol. 217, 503-516[Medline] [Order article via Infotrieve] |
52. | Pai, E. F., Krengel, U., Petsko, G. A., Goody, R. S., Kabsh, W., and Wittinghoffer, A. (1990) EMBO J. 9, 2351-2359[Abstract] |
53. | Sillen, A., Díaz, J. F., and Engelborghs, Y. (2000) Protein Sci. 9, 158-169[Abstract] |
54. |
Lu, C.,
Reedy, M.,
and Erickson, H. P.
(2000)
J. Bacteriol.
182,
164-170 |
55. | Nogales, E., Whittaker, M., Milligan, R. A., and Downing, K. H. (1999) Cell 96, 79-88[Medline] [Order article via Infotrieve] |
56. | Díaz, J. F., Pantos, E., Bordas, E., and Andreu, J. M. (1994) J. Mol. Biol. 238, 214-225[CrossRef][Medline] [Order article via Infotrieve] |
57. | Gigant, B., Curmi, P. A., Martin-Barbey, C., Charbaut, E., Lachkar, S., Lebeau, L., Siavoshian, S., Sobel, A., and Knossow, M. (2000) Cell 102, 809-816[Medline] [Order article via Infotrieve] |
58. | Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef] |