Direct discrimination between models of protein activation by single-molecule force measurements.

Abstract

The limitations imposed on the analyses of complex chemical and biological systems by ensemble averaging can be overcome by single-molecule experiments. Here, we used a single-molecule technique to discriminate between two generally accepted mechanisms of a key biological process--the activation of proteins by molecular effectors. The two mechanisms, namely induced-fit and population-shift, are normally difficult to discriminate by ensemble approaches. As a model, we focused on the interaction between the nuclear transport effector, RanBP1, and two related complexes consisting of the nuclear import receptor, importin beta, and the GDP- or GppNHp-bound forms of the small GTPase, Ran. We found that recognition by the effector proceeds through either an induced-fit or a population-shift mechanism, depending on the substrate, and that the two mechanisms can be differentiated by the data.

Full text

Page 1

Direct Discrimination between Models of Protein Activation by
Single-Molecule Force Measurements
Reinat Nevo,* Vlad Brumfeld,yMichael Elbaum,zPeter Hinterdorfer,§and Ziv Reich*
Departments of *Biological Chemistry,yPlant Sciences, andzMaterials and Interfaces, Weizmann Institute of Science,
Rehovot, Israel; and§Institute for Biophysics, University of Linz, Altenbergerstrs, Linz, Austria
ABSTRACT
be overcome by single-molecule experiments. Here, we used a single-molecule technique to discriminate between two generally
accepted mechanisms of a key biological process—the activation of proteins by molecular effectors. The two mechanisms,
namely induced-fit and population-shift, are normally difficult to discriminate by ensemble approaches. As a model, we focused
on the interaction between the nuclear transport effector, RanBP1, and two related complexes consisting of the nuclear import
receptor, importin b, and the GDP- or GppNHp-bound forms of the small GTPase, Ran. We found that recognition by the effector
proceeds through either an induced-fit or a population-shift mechanism, depending on the substrate, and that the two
mechanisms can be differentiated by the data.
The limitations imposed on the analyses of complex chemical and biological systems by ensemble averaging can
INTRODUCTION
Experimental analysis of complex systems by conventional
methods is often limited by ensemble averaging. This is
particularly problematic for biological systems, where the
size and complexity of the reactants render them highly
inhomogeneous. This inhomogeneity, which persists to the
molecular level and often carries functional importance,
cannot usually be revealed by ensemble approaches. Single-
molecule measurements provide a natural solution to this
problem. Because the molecules are probed one at a time, the
distribution of the molecular properties can be determined
directly rather than inferred, and information hidden in
ensemble-averaged results is unveiled.
A central process in biology is the activation of proteins by
other proteins, protein domains, or small ligands. Signaling
pathways, enzyme activity, and the activation and in-
activation of genes all depend on the switching of proteins
between alternative functional states. The first model for
activation was introduced by Koshland (1958) and was later
extended by Koshland, Nemethy, and Filmer to a sequential
model of allostery (Koshland et al., 1966). In this model,
called induced-fit, the binding of the effector induces a
conformational change in the target protein. The resulting
change in conformation alters the properties of the protein,
and consequently, leads to a change in its activity. The
population-shift model, on the other hand, ascribes changes
in protein activity to a redistribution of pre-existing
conformational isomers. According to this model (known
also as the pre-equilibrium or conformational selection
model), protein structure is regarded as an ensemble of
conformations existing in equilibrium. The ligand binds to
one of these conformations, i.e., the one to which it is most
complementary, thus shifting the equilibrium in favor of
this conformation. This mode of activation is embodied in
the MWC(Monod-Wyman-Changeux)
allostery (Monod et al., 1965). For recent reviews on the
activation of proteins by molecular effectors, see James and
Tawfik (2003) and Kumar et al. (2000).
Presently, there is very little experimental evidence that
distinguishes between the induced-fit and population-shift
models of activation (for notable exceptions, see James et al.,
2003; and Volkman et al., 2001). This is because the
presence of multiple conformations is difficult to ascertain
by conventional methods, even if all conformations are
represented in the ensemble in a significant amount. If the
populations are skewed, an accurate probing of the ensemble
becomes even more problematic. Consequently, there is
a natural tendency to interpret the results as an indication for
an induced-fit, explaining the broad popularity of this model.
In this work, we studied the activation of two related pro-
tein complexes at the single-molecule level, using dynamic
force spectroscopy. We show that the data obtained from the
measurements allow discrimination between the two modes
of activation by comparing the distributions of forces re-
quired to unbind the complexes in the presence and in the
absence of the effector.
model of
MATERIALS AND METHODS
Proteins
His-tagged human importin b was expressed and purified as described in
Nevo et al. (2003). His-tagged Ran (human) was purchased from
Cytoskeleton (Denver, CO) or was obtained from the soluble phase of
Escherichia coli BL21 (DE3; trxB) induced with 0.25 mM IPTG for 8 h, at
30?C. Human RanBP1 was purchased from Cytoskeleton. All proteins were
;95% pure, as determined by silver staining. Loading of Ran with GDP or
GppNHp was made following Nevo et al. (2003). Proteins were analyzed for
Submitted February 24, 2004, and accepted for publication June 22, 2004.
Address reprint requests to Ziv Reich, Dept. of Biological Chemistry,
Weizmann Institute of Science, Rehovot 76100, Israel. Tel.: 972-8-934-
2982; Fax: 972-8-934-6010; E-mail: ziv.reich@weizmann.ac.il.
? 2004 by the Biophysical Society
0006-3495/04/10/2630/05 $2.00
doi: 10.1529/biophysj.104.041889
2630Biophysical JournalVolume 87 October 20042630–2634

Page 2

structural integrity by circular dichroism, and for binding, by ELISA, native
gel electrophoresis, and sizing chromatography.
Tip and surface immobilization
Immobilization of Ran and impb over the AFM tip and the surface (mica)
was made via short polyethylene glycol (PEG) linkers, as described
previously in Nevo et al. (2003).
Dynamic force spectroscopy
Measurements were carried out at room temperature in 50 mM Tris (pH 7.5)/
150 mM NaCl, using a PicoSPM AFM (Molecular Imaging, Phoenix, AZ).
Spring constants of the cantilevers (TM Microscopes, CA) were 0.02–0.03
N/m, as determined by the thermal noise method (Hutter and Bechhoefer,
1993). Force-distance cycles were performed at amplitudes of 100 nm and at
frequencies ranging from 0.2 to 20 Hz; loading rates indicated in the text
were corrected for linkage compliance. The number of rupture events
acquired at each loading rate was between 50 and 700. Specificity of binding
was verified by blocking experiments using free importin b or Ran or by
adding a nonspecific protein (lysozyme; in experiments involving RanBP1).
Data were processed as described in Baumgartner et al. (2000), using Matlab
v6.1. Data shown in the text are presented as Gaussian fits of histograms.
Fitting was carried out using nonlinear least squares analysis made over all
band parameters; r2values were typically .0.95. Analysis of the data
according to their calculated probability density functions gave similar
results.
RESULTS AND DISCUSSION
RanBP1 is a small cytosolic protein that contains a conserved
Ran-binding domain (RanBD). It functions in the disassem-
bly of RanGTP-containing transport complexes that exit
from the nucleus through the nuclear pores by priming these
complexes for GTP hydrolysis, stimulated by the GTPase-
activating protein RanGAP1 (Richards et al., 1995; Bischoff
and Gorlich, 1997; Floer et al., 1997; Kutay et al., 1997,
1998; Lounsbury and Macara, 1997; Gorlich and Kutay,
1999). One such complex is that formed between RanGTP
and the nuclear import receptor importin b1 (impb). Unlike
other transport complexes, efficient dissociation of this
complex requires, in addition to RanBP1, the presence of
importin a—a cargo adaptor of impb (Bischoff and Gorlich,
1997; Floer et al., 1997). In the absence of importin a, the
addition of RanBP1 was actually found to lead to a small
(;3-fold) increase in the association of RanGTP with impb
(Villa Braslavsky et al., 2000). A similar positive, but far
more pronounced, effect is observed when Ran is loaded
with GDP. In contrast to RanGTP, which binds avidly to
impb, RanGDP has an extremely low affinity to the transport
receptor. However, in the presence of RanBP1, stable
trimeric RanBP1-RanGDP-impb complexes are readily
formed (Chi et al., 1996; Villa Braslavsky et al., 2000).
The aforementioned interactions are summarized in Fig. 1,
which shows the results obtained from binding assays of the
three proteins, using native gel electrophoresis. Loaded with
a nonhydrolysable GTP analog (GppNHp), Ran effectively
binds to impb as well as to RanBP1. When mixed together,
the three proteins associate to form a ternary complex in
which Ran is simultaneously bound to impb and RanBP1.
Such a trimeric complex is also formed when impb is
incubated with Ran loaded with GDP in the presence of
RanBP1.
We first address the action of RanBP1 on the complex
formed between impb and RanGppNHp. Using dynamic
force spectroscopy (DFS), we previously showed that this
complex alternates between two conformations of different
adhesion strengths (Nevo et al., 2003). In this methodology
(Fig. 2), which we also applied here, the binding partners are
immobilized onto a cantilevered tip, used in the atomic force
microscope (AFM; Binnig et al., 1986), and to a hard
surface, typically mica. The proteins are immobilized on the
FIGURE 1
presence and absence of RanBP1. Proteins were incubated for 30 min
at room temperature in a reaction buffer containing 50 mM Tris (pH 7.5),
200 mM NaCl, and 1 mM DTT. After incubation, the mixtures were loaded
onto a 10% native polyacrylamide gel. Proteins were present in equimolar
amounts, except RanBP1, which was added in excess.
Binding of impb to GDP- and GppNHp-loaded Ran in the
FIGURE 2
impb were immobilized onto AFM cantilevered tip and mica, respectively,
through short polymer (PEG) linkers as described in Nevo et al. (2003). The
tip was then repeatedly brought to and retracted from the surface, and the
interaction force was measured by following the cantilever deflection, using
the AFM setup. The trace shows a representative force-distance cycle
recorded (at 5 Hz) for RanGDP-impb in the presence of RanBP1.Unbinding
is indicated by the sharp spike in the retraction curve; the parabolic delay
antecedent the spike reflects the extension of the polymer linkers.
Mechanical unbinding of single impb-Ran pairs. Ran and
Protein Activation Studied by DFS2631
Biophysical Journal 87(4) 2630–2634

Page 3

surfaces through short polymer linkers to allow for uncon-
strained recognition and to minimize rebinding of the mole-
cules following rupture (Hinterdorfer et al., 1996). To
achieve single-molecule recognition, both the ligand and the
receptor are immobilized on the surfaces at low densities,
e.g., between 200 and 500 molecules per square micron. For
a typical AFM tip radius of 20–50 nm, these densities
correspond to ;1 molecule per effective tip area. Measure-
ments are conducted by force-distance cycles, where the
cantilever is repeatedly brought to, and retracted from, the
surface. The interaction force is measured by following
the cantilever deflection. This setup allows measurements
of adhesion forces down to the pico-Newton (pN) range
and detection of states with a fractional occupancy as low as
a few percent.
Owing to its two bound states, RanGppNHp-impb gives
rise to unique, bimodal distributions of unbinding forces
(Nevo et al., 2003). The dashed lines in Fig. 3 A show one
such distribution. The two states of the complex are
represented by two partially overlapping force populations,
of which the higher-strength population is ;21/2times larger
than the lower-strength population. This situation is reversed
when RanBP1 is added to the solution after which the
higher-strength population is diminished and the lower-
strength population predominates the ensemble (Fig. 3 A,
solid lines). Note that although the relative size of the two
force populations changes upon addition of the effector, their
means do not. As a control, we replaced RanBP1 with
lysozyme. The results were similar to those obtained for the
complex in the absence of the RanBP1.
Next, we investigated the effect of RanBP1 on the
RanGppNHp-impb interaction over a broad range of loading
rates. This is a common practice in force spectroscopy
measurements since the measured forces are not only
contingent on the molecules themselves, but also depend
on the loading rate rf(¼ Df/Dt) at which force is applied to
the complex (Evans and Ritchie, 1997; Heymann and
Grubmuller, 2000; Evans, 2001; Dudko et al., 2003).
Specifically, the most probable force for unbinding f* (taken
as the maximum of the force distributions) is related to the
loading rate through f* ? fbln(rftoff/fb), where the force scale
fbis given by the ratio of thermal energy kBT to a small
(,1 nm) molecular length xb, which marks the thermally
averaged projection of the transition state along the direction
of the force. Alteration of the loading rate was achieved by
varying the speed of retraction, giving rise to loading rates
spanning almost three orders of magnitude in scale. The
results are shown in terms of the most probable force for
unbinding, plotted against the logarithm of the loading rate.
In the absence of RanBP1, the strength spectrum of
RanGppNHp-impb is characterized by two well-separated
f* versus log(rf) curves corresponding to energy barriers
encoded in the lower-strength (bottom) and higher-strength
(top) conformations of the complex (Fig. 4 A, dashed lines;
Nevo et al., 2003). The addition of RanBP1 greatly
diminished contributions from the high-strength conforma-
tion throughout the whole range of loading rates, leaving
a small number of unbinding events recorded for this
conformation (Fig. 3 C). The other, now largely predom-
inating population, gave rise to a strength spectrum that was
practically identical to the one produced by the low-strength
conformation of the complex in the absence of the effector
(Fig. 4 A, solid line). These results clearly support a model in
which activation of RanGppNHp-impb is achieved by
a dynamic shift between its two conformations. They also
indicate that, in addition to its previously reported ability to
promote association between RanGppNHp and impb (Villa
Braslavsky et al., 2000), RanBP1 also facilitates their disso-
ciation by shifting the equilibrium toward the low-strength
conformation of the complex.
We now turn to the effect of RanBP1 on the interaction
between impb and RanGDP. This form of Ran has a very
low affinity to impb. Nevertheless, binding can still be
detected by force spectroscopy measurements, which show
that RanGDP associates weakly with impb to form a single
bound state characterized by unimodal distributions of
rupture forces (Figs. 3 B and 4 B, dashed lines; Nevo et al.,
2003). In contrast to its effect on RanGppNHp-impb,
RanBP1 led to a marked shift of the distributions obtained
for RanGDP-impb to higher unbinding forces throughout the
FIGURE 3
RanGDP-impb complexes in the absence (dashed lines) and presence (solid
line) of RanBP1. (A and B) Distributions of unbinding forces recorded at
20 Hz. Data are presented as Gaussian fits of histograms; the width of the
bins represents the thermal noiseof the cantilever. Forclarity, histogramsare
shown only for Ran-impb pairs unbound in the presence of RanBP1. In this
and the following figure, data shown for pairs dissociated in the absence of
RanBP1 (dashed lines) were taken from Nevo et al. (2003). The marked
increase in the number of unbinding events recorded for RanGDP-impb in
the presence of RanBP1 reflects the ability of the latter to facilitate
association of RanGDP with impb (Villa Braslavsky et al., 2000, and see
text). (C) Unbinding force distributions recorded for RanGppNHp-impb in
the presence of RanBP1 at different loading rates. Albeit significantly
skewedtoward the low-strengthconformation,the ensemblestill revealstwo
populations (t-test p-value, 3 3 10?5).
Unbinding force distributions of RanGppNHp-impb and
2632Nevo et al.
Biophysical Journal 87(4) 2630–2634

Page 4

whole range of loading rates (Figs. 3 B and 4 B, solid lines).
Such a behavior is expected for an induced-fit mechanism,
where the binding of the effector induces the formation of
a new structure in the complex, which results in higher
stability.
The results obtained in this work demonstrate that single-
molecule measurements can effectively discriminate be-
tween induced-fit and population-shift mechanisms of
activation. The major limitation appears to be the inability
to detect states with very low fractional occupancy (for the
technique used here—lower than a few percent). In addition,
the two binding mechanisms may coexist or occur conse-
cutively (i.e., population-shift followed by local structural
rearrangements). In such cases, only the predominant
mechanism is likely to be exposed. These limitations,
however, also exist in bulk approaches, which are further
impaired by ensemble-averaging effects.
Within the limitations discussed above, our data indicate
that recognition of RanGDP-impb by RanBP1 proceeds by
an induced-fit process. The exact nature of the structural
rearrangements triggered in RanGDP by RanBP1 is pre-
sently unknown, since the crystal structure of the complex is
not available (for the structure of RanGppNHp-impb, see
Vetter et al., 1999a). One region in RanGDP that probably
undergoes a large conformational change upon RanBP1
binding is the C-terminal region of Ran that serves as a major
recognition site for RanBP1, but is thought to dock to a basic
patch in Ran (Vetter et al., 1999b). [In RanGppNHp, the
C terminus is probably displaced, since it is more exposed
and appears to be bound less tightly to the rest of the protein
(Vetter et al., 1999a,b, and references therein)]. The
displacement of Ran’s C-terminus by RanBP1 is believed
to greatly facilitate association of RanGDP with impb (Villa
Braslavsky et al., 2000). This effect is indeed reflected by the
significant increase in the number of rupture events recorded
for the pair in the presence of the effector (Fig. 3 B). How-
ever, structural changes in regions outside the C-terminal
region of Ran are probably necessary to account for the pro-
nounced effect of RanBP1 on the dissociation of RanGDP
from impb.
On the other hand, the results obtained for the complex
between impb and RanGppNHp fit very well to a model
based on a dynamic shift between pre-existing alternative
conformations. According to this model, RanBP1 binds to
the lower-strength conformation of RanGppNHp-impb, thus
shifting the equilibrium in favor of this conformation. A
similar two-state allosteric behavior was demonstrated for
the bacterial response regulator NtrC (Volkman et al., 2001)
and calmodulin (Malmendal et al., 1999). Recently, a
population selection model has been suggested for the
recognition of the impb-binding (IBB) domain of importin
a by impb (Koerner et al., 2003).
How RanBP1 actually distinguishes between the two
conformations of the RanGppNHp-impb complex is not
clear at present. P-NMR studies have shown that RanGTP
alternates between two conformations, of which only one
could be detected in the presence of RanBP1 (Geyer et al.,
1999). Although this effect was not obtained when Ran was
loaded by GppNHp, it was observed for GppNHp-loaded
Ras (Geyer et al., 1996), which is closely related to Ran. A
potential target for discrimination is the basic patch of Ran,
which interacts with an acidic loop connecting two centrally
located helices of impb. The interface formed between Ran’s
basic patch and the acidic loop of impb may be accessible to
an N-terminal extension present in RanBP1 or in the Ran-
binding domains of RanBP2 (Vetter et al., 1999a). It has
been suggested that the N-terminus of the Ran-binding
domains, which is acidic, competes with the acidic loop of
impb for the basic patch of Ran (Vetter et al., 1999a; Villa
Braslavsky et al., 2000). If this prediction is correct, then the
two conformations of RanGppNHp-impb may differ in their
accessibility to the N-terminus of RanBP1, with the lower-
strength conformation providing better access to the basic
patch.
We thank H. Gruber and C. Riener for the synthesis of the PEG linkers and
O. Bogin for his help in the expression of Ran and impb. We also thank M.
Fainzilber, R. Kapon, F. Kienberger, C. Stroh, A. Topchik, E. Wachtel, and
the anonymous reviewers for helpful comments and suggestions.
FIGURE 4
absence of RanBP1. Most probable unbinding forces were plotted against
the logarithm of the loading rate. A and B show the force spectra obtained for
RanGppNHp-impb and RanGDP-impb in the absence (dashed lines) and
presence (solid lines) of RanBP1. Error bars represent standard deviation.
Force spectra of Ran-impb complexes in the presence and
Protein Activation Studied by DFS 2633
Biophysical Journal 87(4) 2630–2634

Page 5

This work was supported by the Charles H. Revson Foundation (Israel
Science Foundation) and the Austrian National Science Foundation.
REFERENCES
Baumgartner, W., P. Hinterdorfer, and H. Schindler. 2000. Data analysis of
interaction forces measured with the atomic force microscope. Ultra-
microscopy. 82:85–95.
Binnig, G., C. F. Quate, and C. Gerber. 1986. Atomic force microscope.
Phys. Rev. Lett. 56:930–933.
Bischoff, F. R., and D. Gorlich. 1997. RanBP1 is crucial for the release of
RanGTP from importin beta- related nuclear transport factors. FEBS Lett.
419:249–254.
Chi, N. C., E. J. Adam, G. D. Visser, and S. A. Adam. 1996. RanBP1
stabilizes the interaction of Ran with p97 nuclear protein import. J. Cell
Biol. 135:559–569.
Dudko, O. K., A. E. Filippov, J. Klafter, and M. Urbakh. 2003. Beyond the
conventional description of dynamic force spectroscopy of adhesion
bonds. Proc. Natl. Acad. Sci. USA. 100:11378–11381.
Evans, E. 2001. Probing the relation between force—lifetime—and
chemistry in single molecular bonds. Annu. Rev. Biophys. Biomol.
Struct. 30:105–128.
Evans, E., and K. Ritchie. 1997. Dynamic strength of molecular adhesion
bonds. Biophys. J. 72:1541–1555.
Floer, M., G. Blobel, and M. Rexach. 1997. Disassembly of RanGTP-
karyopherin beta complex, an intermediate in nuclear protein import.
J. Biol. Chem. 272:19538–19546.
Geyer, M., R. Assheuer, C. Klebe, J. Kuhlmann, J. Becker, A.
Wittinghofer, and H. R. Kalbitzer. 1999. Conformational states of the
nuclear GTP-binding protein Ran and its complexes with the exchange
factor RCC1 and the effector protein RanBP1. Biochemistry. 38:11250–
11260.
Geyer, M., T. Schweins, C. Herrmann, T. Prisner, A. Wittinghofer, and
H. R. Kalbitzer. 1996. Conformational transitions in p21ras and in its
complexes with the effector protein Raf-RBD and the GTPase activating
protein GAP. Biochemistry. 35:10308–10320.
Gorlich, D., and U. Kutay. 1999. Transport between the cell nucleus and the
cytoplasm. Annu. Rev. Cell Dev. Biol. 15:607–660.
Heymann, B., and H. Grubmuller. 2000. Dynamic force spectroscopy of
molecular adhesion bonds. Phys. Rev. Lett. 84:6126–6129.
Hinterdorfer, P., W. Baumgartner, H. J. Gruber, K. Schilcher, and H.
Schindler. 1996. Detection and localization of individual antibody-
antigen recognition events by atomic force microscopy. Proc. Natl. Acad.
Sci. USA. 93:3477–3481.
Hutter, J. L., and J. Bechhoefer. 1993. Calibration of atomic-force
microscope Tips. Rev. Sci. Instrum. 64:1868–1873.
James, L. C., P. Roversi, and D. S. Tawfik. 2003. Antibody multispecificity
mediated by conformational diversity. Science. 299:1362–1367.
James, L. C., and D. S. Tawfik. 2003. Conformational diversity and protein
evolution—a 60-year-old hypothesis revisited. Trends Biochem. Sci.
28:361–368.
Koerner, C., T. Guan, L. Gerace, and G. Cingolani. 2003. Synergy of silent
and hot spot mutations in importin beta reveals a dynamic mechanism for
recognition of a nuclear localization signal. J. Biol. Chem. 278:16216–
16221.
Koshland, D. E., Jr., G. Nemethy, and D. Filmer. 1966. Comparison of
experimental binding data and theoretical models in proteins containing
subunits. Biochemistry. 5:365–385.
Koshland, D. E. J. 1958. Application of a theory of enzyme specificity to
protein synthesis. Proc. Natl. Acad. Sci. USA. 44:98–123.
Kumar, S., B. Ma, C. J. Tsai, N. Sinha, and R. Nussinov. 2000. Folding and
binding cascades: dynamic landscapes and population shifts. Protein Sci.
9:10–19.
Kutay, U., F. R. Bischoff, S. Kostka, R. Kraft, and D. Gorlich. 1997. Export
of importin a from the nucleus is mediated by a specific nuclear transport
factor. Cell. 90:1061–1071.
Kutay, U., G. Lipowsky, E. Izaurralde, F. R. Bischoff, P. Schwarzmaier, E.
Hartmann, and D. Gorlich. 1998. Identification of a tRNA-specific
nuclear export receptor. Molecular. Cell. 1:359–369.
Lounsbury, K. M., and I. G. Macara. 1997. Ran-binding protein 1
(RanBP1) forms a ternary complex with Ran and karyopherin-b and
reduces Ran GTPase-activating protein (RanGAP) inhibition by
karyopherin-b. J. Biol. Chem. 272:551–555.
Malmendal, A., J. Evenas, S. Forsen, and M. Akke. 1999. Structural
dynamics in the C-terminal domain of calmodulin at low calcium levels.
J. Mol. Biol. 293:883–899.
Monod, J., J. Wyman, and J. P. Changeux. 1965. On the nature of allosteric
transitions: A plausible model. J. Mol. Biol. 12:88–118.
Nevo, R., C. Stroh, F. Kienberger, D. Kaftan, V. Brumfeld, M. Elbaum, Z.
Reich, and P. Hinterdorfer. 2003. A molecular switch between alternative
conformational states in the complex of Ran and importin b1. Nat.
Struct. Biol. 10:553–557.
Richards, S. A., K. M. Lounsbury, and I. G. Macara. 1995. The C-terminus
of the nuclear RAN/TC4 GTPase stabilizes the GDP-bound state and
mediates interactions with RCC1, RAN-GAP, and HTF9A/RANBP1.
J. Biol. Chem. 270:14405–14411.
Vetter, I. R., A. Arndt, U. Kutay, D. Gorlich, and A. Wittinghofer. 1999a.
Structural view of the Ran-Importin b interaction at 2.3 A resolution.
Cell. 97:635–646.
Vetter, I. R., C. Nowak, T. Nishimoto, J. Kuhlmann, and A. Wittinghofer.
1999b. Structure of a Ran-binding domain complexed with Ran bound to
a GTP analogue: Implications for nuclear transport. Nature. 398:39–46.
Villa Braslavsky, C. I., C. Nowak, D. Gorlich, A. Wittinghofer, and J.
Kuhlmann. 2000. Different structural and kinetic requirements for the
interaction of Ran with the Ran-binding domains from RanBP2 and
importin-b. Biochemistry. 39:11629–11639.
Volkman, B. F., D. Lipson, D. E. Wemmer, and D. Kern. 2001. Two-state
allosteric behavior in a single-domain signaling protein. Science.
291:2429–2433.
2634Nevo et al.
Biophysical Journal 87(4) 2630–2634