doi:10.1006/jmbi.2000.4262 available online at http://www.idealibrary.com on
J. Mol. Biol. (2001) 305, 121±135
Structure-activity Relationships in Flexible Protein
Domains: Regulation of rho GTPases by RhoGDI and
D4 GDI
Alexander P. Golovanov1, Tsung-Hsien Chuang2
Celine DerMardirossian2, Igor Barsukov1, Dawn Hawkins1
Ramin Badii1, Gary M. Bokoch2*, Lu-Yun Lian1* and
Gordon C. K. Roberts1
1
Department of Biochemistry
and Biological NMR Centre
University of Leicester
University Road, Leicester
LE1 7RH, UK
2
Departments of Immunology
and Cell Biology, The Scripps
Clinic and Research Institute
10550 N. Torrey Pines Road
La Jolla, CA 92037, USA
The guanine dissociation inhibitors RhoGDI and D4GDI inhibit guanosine 50 -diphosphate dissociation from Rho GTPases, keeping these small
GTPases in an inactive state. The GDIs are made up of two domains: a
¯exible N-terminal domain of about 70 amino acid residues and a folded
134-residue C-terminal domain. Here, we characterize the conformation
of the N-terminal regions of both RhoGDI and D4GDI using a series of
NMR experiments which include 15N relaxation and amide solvent accessibility measurements. In each protein, two regions with tendencies to
form helices are identi®ed: residues 36 to 58 and 9 to 20 in RhoGDI, and
residues 36 to 57 and 20 to 25 in D4GDI. To examine the functional roles
of the N-terminal domain of RhoGDI, in vitro and in vivo functional
assays have been carried out with N-terminally truncated proteins. These
studies show that the ®rst 30 amino acid residues are not required for
inhibition of GDP dissociation but appear to be important for GTP
hydrolysis, whilst removal of the ®rst 41 residues completely abolish the
ability of RhoGDI to inhibit GDP dissociation. The combination of structural and functional studies allows us to explain why RhoGDI
and D4GDI are able to interact in similar ways with the guanosine
50 -diphosphate-bound GTPase, but differ in their ability to regulate
GTP-bound forms; these functional differences are attributed to the
conformational differences of the N-terminal domains of the guanosine
50 -diphosphate dissociation inhibitors. Therefore, the two transient
helices, appear to be associated with different biological effects of
RhoGDI, providing a clear example of structure-activity relationships in a
¯exible protein domain.
# 2001 Academic Press
*Corresponding authors
Keywords: dissociation inhibitor; GTPase; Rac1; RhoGDI; D4/LyGDI
Introduction
The function of a protein is, of course, generally
assumed to be closely linked to its three-dimen-
sional structure. Many globular proteins contain
local segments or loops which are disordered;
however, there is increasing evidence for the widespread existence of proteins or domains which are
Present addresses: A.P.G. and L.-Y.L., Department of Biomolecular Sciences, University of Manchester Institute of
Science and Technology, P.O. Box 88, Manchester M60 1QD, UK.
A.P.G. and T.H.C. have contributed equally to this work.
E-mail addresses of the corresponding authors: bokoch@scripps.edu; lu-yun.lian@umist.ac.uk
Abbreviations used: CSI, Chemical shift index; GDI, GDP-dissociation inhibitor; GDP, guanosine 50 -diphosphate;
HMQC, heteronuclear multiple-quantum correlation; HSQC, heteronuclear single-quantum correlation; NOE, nuclear
Overhauser effect; hyTEMPO, 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy; TROSY, transverse relaxation-optimized
spectroscopy; GTPase, GTP hydrolyzing protein; GEF, guanosine nucleotide exchange factor; GAP, GTPase-activating
proteins.
0022-2836/01/010121±15 $35.00/0
# 2001 Academic Press
122
``unfolded'' in solution under physiological conditions.1 Examples include DNA-binding domains,
transcription activation domains, proteins involved
in transcription initiation, proteins of the membrane fusion SNARE complex, the cyclin dependent kinase inhibitor p21waf1/Cip1, and a
®bronectin-binding protein.1 ± 8 The function of
these proteins involves interaction with other proteins or nucleic acids, and this is associated with a
transition to a folded state; the requirement for this
transition, driven by the binding energy, may be
an important contribution to the speci®city of the
interaction.1,7 The apparently unfolded proteins or
domains often contain regions with transient secondary structures, which although dif®cult to
observe or detect, are important in that they form
the sites for productive interactions with target
molecules. It is therefore important to be able to
characterize the unfolded states of these proteins
or domains, and NMR spectroscopy has proved to
be very valuable in this context.9,10 Here, we
describe a comparison of the structure-activity
relationships of one such ¯exible domain, the
N-terminal domain from RhoGDI and D4GDI
which are proteins which regulate rho family GTPhydrolyzing proteins (GTPases).
The rho family of small GTPases which includes
the isoforms of rho, Rac1, Cdc42 and TC10 are
important regulators of cell function. They have
been implicated in the control of cell motility,
adhesion, cytokinesis, pinocytosis, endocytosis,
secretion, axonal outgrowth, growth arrest and cell
death, as well as cell cycle progression and transformation.11 As for other members of the ras superfamily of GTPases, the cycling of these proteins
between the GTP-bound (``active'') and GDPbound (``inactive'') forms is controlled by guanine
nucleotide exchange factors (GEFs), which catalyse
the exchange of GTP for GDP, and GTPase-activating proteins (GAPs), which accelerate GTP hydrolysis.12,13 In addition, the activity of rho family
GTPases is controlled by guanine nucleotide dissociation inhibitors, the Rho guanosine 50 -phosphate dissociation inhibitors (GDIs).14,15 Three
GDIs have been found, termed RhoGDI (RhoGDI1), D4GDI (RhoGDI-2 or LyGDI) and RhoGDIg
(RhoGDI-3). RhoGDI is ubiquitously expressed,15
while D4GDI is found in haematopoetic cells;16,17
both have a broad range of activity toward the rho
proteins. RhoGDI-318 is expressed predominantly
in the brain, lung and pancreas, and is speci®c for
rhoB and rhoG; unlike the other two cytoplasmic
forms, RhoGDI-3 is associated with the membrane
or possibly the cytoskeleton.
RhoGDI, the best characterized GDI, exhibits
three distinct biochemical functions: (a) inhibition
of guanosines-disphosphate (GDP) dissociation,
which keeps the rho guanosine triphosphate
hydrolyzing proteins (GTPases) in an inactive
state, (b) inhibition of intrinsic or GAP-stimulated
GTP hydrolysis, which maintains the rho GTPases
in an active state, and (c) control of the partitioning
of the GTPase between cytosol and membrane.
Structure and Activity of Flexible Protein Domains
D4GDI and RhoGDI3 have both been shown to
inhibit GDP dissociation as well, but have not been
evaluated as inhibitors of GTP hydrolysis. The
physiological role of the interaction of RhoGDI
with the GTP-bound form of a GTPase remains to
be established. Overall, when exogenously introduced into cells the GDIs behave as negative regulators by maintaining rho GTPases as inactive
cytosolic forms which are unable to effectively
interact with GEFs and/or downstream target molecules. However, in some situations, for example,
ezrin/radixin/moesin19 and PI 5-kinase,20 the
GTPase has been reported to bind to an effector
molecule as a complex with RhoGDI, suggesting
that RhoGDI may also play a role in directing the
GTPase to the effector. In unstimulated cells, the
majority of the rho family GTPases appear to exist
in the cytoplasm as a complex with RhoGDI.21,22
We and others have previously shown that
RhoGDI is made up of two domains: a ¯exible
N-terminal domain (residues 1 to 69) and a
C-terminal domain (70 to 204) which adopts an
immunoglobulin-like fold.23 ± 25 The N-terminal
domain is essential for the binding of RhoGDI to
Rac1 and Cdc42.23,24 The recent structures of the
RhoA-RhoGDI,26 Cdc42-RhoGDI,27 and Rac2D4GDI28 show that the ¯exible N-terminal domain
is the regulatory arm of the GDIs which, in part,
becomes ordered in the complex, in agreement
with previous NMR studies.25
The observation that the formation of the complex between RhoGDI and the GTPase involves a
marked ordering of the N-terminal domain of the
GDI means that an understanding of the structurefunction relationships of this key domain requires
a detailed knowledge of its conformational characteristics in the uncomplexed as well as the complexed state. No structure of an uncomplexed fulllength RhoGDI has so far been reported. We have
now undertaken a detailed conformational characterization of the N-terminal domains of RhoGDI
and D4GDI, using a range of NMR techniques
which allow us to detect transient helical structures
which are preserved and stabilized in the GTPase
complex. We show that, whilst both GDIs interact
in similar ways with the GDP-bound GTPase, they
differ markedly in their ability to regulate GTPbound forms; we further demonstrate that these
functional differences can be attributed to differences in the structure of N-terminal domain of the
two GDIs.
Results
Flexible domains of GDIs have incipient
helical structures
In order to analyse the conformational characteristics of the ¯exible domain of RhoGDI and D4GDI
in details, several NMR approaches were used,
each sensitive to different aspects of the structure:
analysis of intensities of cross-peaks in nuclear
Overhauser effect (NOE) spectra, which re¯ect
123
Structure and Activity of Flexible Protein Domains
inter-proton distances, and the 13C chemical
shift index (CSI)29 both provide information on
secondary structure; the effects of the paramagnetic
relaxation probe 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (hyTEMPO) on the longitudinal
relaxation rates of amide protons allows one to
assess accessibility of individual residues to the
probe; and the analysis of heteronuclear 15N-relaxation data provides information on the rapid
motions of amino acid residues. Each of these
approaches is described in turn below.
NOE and CSI data: secondary structure
We have previously reported that in RhoGDI a
short stretch of residues in the ¯exible domain, 4858, has a tendency to form a helical structure.25 A
closer inspection of intra and inter-residue NOEs
contacts, particularly NH-NH NOEs in spectra
resolved in both the 1H dimension nuclear
Overhauser effect heteronuclear single quantum
correlation, (NOESY-HSQC (denoted by d(H)NN)
and in the 15N-dimension heteronuclear multiplequantum
correlation,
HMQC-NOESY-HSQC
(denoted by d(N)NN) reveals other regions with
weaker helix-forming tendencies (Figure 1(a)).
Combining the NOE and CSI information for
RhoGDI, it appears that regions 9-20 and 37-43
also contain incipient helical structures. These
observations are consistent with the helical propensities predicted for the N-terminal domain using
AGADIR,30 which predicts up to 60 % of helical
structure content for the region 45-56, but lower
percentages for regions 9-14, 35-44 and 57-61
(Figure 1(a)). For D4GDI{ the agreement between
the NOE and CSI data is less clear; the NH-NH
NOEs indicate some helical structure in the regions
47-59 and 21-27, while the CSI suggests the
presence of helices in the regions 45-57, 38-41 and
20-25 (Figure 1(b)). AGADIR predicts the existence
of transient helices in the regions 45-56, 19-25 and
35-44; therefore the combined NOE and CSI data
agree well with the prediction.
(unpublished data). In the range of hyTEMPO concentration used in the current studies (up to 8 mM)
no noticeable signal broadening was observed for
the residues from the ¯exible N-terminal domain.
Figure 2(a) reveals the regions on RhoGDI and
D4GDI in which the amides are relatively accessible to the probe. In both RhoGDI and D4GDI, residues 28 to 35 and 57 to 64 are more exposed than
other residues in the N-terminal domain, and we
suggest that these stretches of polypeptide chain
form ¯exible loops. By contrast, several continuous
stretches of residues, 9 to 24 and 40 to 56 in
RhoGDI and 13 to 25 and 36 to 56 in D4GDI, show
lesser effects of hyTEMPO, indicating that they are
more shielded than average from the probe; these
regions correspond well with the helical regions
described above. As a comparison, the relaxation
rates of some of the residues from C-terminal
domain of RhoGDI, which are completely buried
in the hydrophobic core, do not change upon
addition of hyTEMPO (i.e. RTEMPO ˆ 0), providing
further evidence that the regions of transient helical structure located at the N-terminal domain are
not completely shielded from the paramagnetic
probe.
There are several advantages in using 2H
enriched (ca 75 %) 15N-labelled samples of RhoGDI
and D4GDI for the non-selective inversion-recovery experiments. A substantial decrease in the line
widths, and hence improved resolution and signalto-noise ratio, relative to the undeuterated protein
were obtained, together with a signi®cantly
decreased cross-relaxation between protons, the
latter usually leading to similar relaxation rates
being observed throughout the sample. Because
there are fewer spins positioned at increased distance, it is possible to observe a wide range of
relaxation rates for the amide protons in a 2H
enriched protein. The relaxation of these amide
protons is also more sensitive to the relaxation
properties of the solvent.32
15
N relaxation
15
Paramagnetic effect on longitudinal relaxation rates
The enhancement of the paramagnetic longitudinal relaxation rate of amide protons in the presence
of the soluble relaxation probe hyTEMPO was
used as the measure of solvent exposure of amides.
Concentrational dependence of the longitudinal
relaxation rates (see Materials and Methods) were
measured using inversion-recovery version of
1
H-15N transverse relaxation-optimized spectroscopy (TROSY),31 as this type of experiment
provided spectra with very good resolution for
both the uncomplexed and complexed GDIs
{ For convenience, the residue numbering refers to the
RhoGDI sequence; for example, in the text residue 60 of
D4GDI is in fact residue 57 in the actual D4GDI
sequence (see sequence alignment in Figure 4a).
N relaxation parameters (NOEs, relaxation
rates R1 and R2) re¯ect the dynamics of the polypeptide backbone9,33 (Figure 2(b)). Of these parameters, the heteronuclear NOE is probably the
most useful for the qualitative description of mobility at the individual residue level: for rigid parts
of proteins, NOEs are positive and close to 0.8,
while for extremely ¯exible parts of proteins,
NOEs are negative. The average heteronuclear
NOE values for the N-terminal region of both
GDIs are indicative of high mobility. Closer examination of the measured NOEs within this region
of RhoGDI and D4GDI (Figure 2(b)) reveals clear
trends which are consistent with the data on secondary structure and accessibility described above.
Both GDIs have restricted mobility in region 43-57,
corresponding to the position of the major transient helix, although there are differences in the
dynamics of this region. The higher values of the
124
Structure and Activity of Flexible Protein Domains
Figure 1. Secondary structures in the N-terminal domains of (a) RhoGDI and (b) D4GDI. The data includes helical
content predicted by the AGADIR program;30 and consensus chemical shift indexes (CSI).29 Sequential and intra-residue NOE cross-peak intensities obtained from 3D spectra (classi®ed as strong, medium and weak) are represented by
the height of the bars. Asterisks indicate NOEs not observed because of signal overlap, gaps indicate absence of NOE
cross-peaks. d(H)NN and d(N)NN refer to cross-peaks observed in 3D 1H-15N NOESY-HSQC and HMQC-NOESYHSQC spectra, respectively (see Methods).
NOEs for RhoGDI (average NOE ˆ 0.35(0.13))
than in D4GDI (average NOE ˆ 0.20(0.08)), are
compatible with the greater helical content for this
helix in RhoGDI predicted by AGADIR. Following
this helix, both GDIs show negative NOEs, indicative of high mobility (residues 60 to 63 in RhoGDI
and 57 to 64 in D4GDI), corresponding to the
regions accessible to hyTEMPO. The major differences between the two GDIs found in the most Nterminal part of the protein are: in RhoGDI negative NOEs are observed for residues 4 to 7 and 28
to 36 with restricted mobility indicated for residues
8 to 27; in D4GDI the most mobile residues are 5
to 22, and 28, with restricted mobility of residues
23 to 27, and 32 to 38. The differences between
RhoGDI and D4GDI are also clearly manifested by
the variations in the R2 values as a function of
sequence, which are very similar to those of the
heteronuclear NOE values (Figure 2(b)).
Broadly, there are two ways in which dynamic
information can be extracted from the measured
relaxation parameters. The ®rst, the so-called
``model-free'' approach,34 makes assumptions
about isotropic tumbling of the molecules and the
number and magnitude of the correlation times for
internal motion. However, ¯exible and unstructured proteins undergo very complex motions and
the assumptions made in the model-free approach
are not necessarily valid. Various modi®cations of
the model-free analysis have been used in attempts
to describe more complex motions by introducing
extra parameters, although additional experimental
information, such as relaxation data at various
®eld strengths, is then required. A second and
more general approach is to extract the dynamical
information directly from spectral density functions, J(o), which represent the frequency distribution of rotational motions of N-H bond vectors
and provide an indication about characteristic
timescales of these motions.35-37 This approach
makes no assumptions about the motions to be
investigated, and hence is valid for ¯exible
domains, and also requires a minimal number of
experimental parameters. It is very valuable for
direct comparisons of the timescales of motions
between different proteins or different parts of the
same protein.
Using the reduced spectral density function
approach35-37 (see Materials and Methods for the
formulae used) and the heteronuclear relaxation
data obtained at 500 MHz (proton frequency), J(o)
can be sampled at three different frequencies: 0,
oN ˆ 50.6 MHz and oH ˆ 500 MHz (i.e. J(0), J(N)
and J(H)). Since the area under J(o) is normalized,
the presence of high-frequency motions (faster than
the tumbling of the protein 108 sÿ1) leads to
lower values of J(0), and higher values of J(H). The
N-terminal domains of RhoGDI and D4GDI show,
on average, enhanced mobility (reduced J(0) and
higher J(H) values) relative to the folded domain
Structure and Activity of Flexible Protein Domains
125
Figure 2. Relaxation data for the N-terminal domain of RhoGDI (*, left column) and D4GDI (&, right column) at
288 K. For some residues error bars are smaller than the symbols. (a) Paramagnetic effect on longitudinal relaxation
rates of amide protons measured in the presence of the relaxation agent hyTEMPO: residues 28 to 35 and 57 to 64
are more exposed than other residues in the N-terminal domain. Residues 9 to 24 and 40 to 56 in RhoGDI and 14 to
24 and 36 to 56 in D4GDI show lesser effects of hyTEMPO, indicating that they are more shielded from the relaxation
probe. (b) 15N relaxation rates R1, R2 and NOE data: The measured NOEs for RhoGDI and D4GDI reveal trends
which are consistent with the data on secondary structure and accessibility for this N-terminal domain. Both GDIs
have restricted mobility in region 43-57, corresponding to the position of the major transient helix, which has been
located between residues 48-58 (RhoGDI) and 46-55 (D4GDI) based on the CSI and 1H-1H NOE data. (c) Reduced
spectral density functions J(0), J(50) and J(500): High-frequency motions can be observed for the ®rst few residues at
the N-termini both of RhoGDI and D4GDI, for residues 58 to 63 of RhoGDI and 57 to 64 of D4GDI. Slower motions
are observed for residues which are either close to the folded domain or are in the ``helix-forming'' regions; these
include residues 66 to 67, residues 46 to 56 of RhoGDI and 46 to 54 of D4GDI.
on the nanoseconds to picoseconds timescale.38
Within the N-terminal domain of the GDIs, slower
motions (in nanosecond timescale) are observed for
residues which are either close to the folded
domain or are in the ``helix-forming'' regions
(Figure 2(c)); these include residues 66 to 67, residues 46 to 56 of RhoGDI and 46 to 54 of D4GDI,
and to a lesser extent, residues 36 to 45 in both
proteins. Although the region 46-56 shows slow
mobility in both RhoGDI and D4GDI, the values of
J(0) and J(H) in D4GDI relative to those in RhoGDI
are consistent with this helix being more ``persistent'' in the latter protein than in D4GDI. The
dependence of J(0) on residue number is quite
different for RhoGDI and D4GDI at the extreme
N termini: for RhoGDI J(0) increases gradually
126
from the N terminus up to residue 9, plateauing
out from this residue onwards to residue 45, whilst
in D4GDI, J(0) increases steadily up to residue 22,
and again plateauing out until residue 45,
suggesting that the extreme N-terminal region of
RhoGDI undergoes slower motions than that of
D4GDI. The values of J(H) indicate that in D4GDI
a longer stretch of N-terminal residues is involved
in fast motions (on the nanosecond-picosecond
timescale) than in RhoGDI (Figure 2(c)). Other
regions possessing fast motions are residues 31 to
34 and 58 to 63 of RhoGDI and 57 to 64 of D4GDI,
corresponding to the exposed ¯exible loops, which
were identi®ed by hyTEMPO experiments.
The values of experimental heteronuclear relaxation parameters for RhoGDI and D4GDI, as well
as the values of spectral density functions, are comparable to those of other proteins possessing ¯exible segments or domains.39,40 The pro®les shown
in Figure 2(b) and (c) are very similar to the corresponding pro®les of the ¯exible N terminus of the
basic leucine-zipper domain of the yeast transcription factor GCN4, which exists as an ensemble of
transiently formed helical structures in free state,
and achieves a stable structure when bound to
DNA.40 In particular, for the transcription factor,
J(N) also has lower values both for the ®rst few
residues of the N terminus (involved in faster
motions on picosecond timescale), and for the
well-structured part of the protein (involved in
slower motions in nanosecond time scale), whereas
the transient helical region in the middle has higher values of J(N).
In summary, in both RhoGDI and D4GDI the
region 36-57 has a clear incipient helical structure,
the helix apparently being more populated in
RhoGDI than in D4GDI. Each of the GDIs also
appears to have another region at the N terminus
which has a weaker tendency to form a helix,
although the position of these regions differs in the
Structure and Activity of Flexible Protein Domains
two proteins: residues 9 to 20 for RhoGDI and 20
to 25 for D4GDI.
Identification of regions of the N-terminal
domain involved in GTPase binding
Addition of equimolar amounts of non-isoprenylated unlabeled Rac1 to 15N,2H-labelled D4GDI
caused signi®cant changes in chemical shifts in the
1
H-15N TROSY31 spectrum, re¯ecting one-to-one
complex formation (in slow exchange on the NMR
timescale). Similar observations were made previously for RhoGDI-Rac1 complex.23,25 The spectra
of RhoGDI and D4GDI in the free and bound
states are shown in Figure 3. A high threshold is
chosen for plotting the spectra to show only the
signals with high intensities and narrower line
widths, which arise from the ¯exible N-terminal
domains of the proteins.
Residues in the N-terminal region of D4GDI
whose chemical shifts were signi®cantly affected
by Rac1 binding were identi®ed using the minimum chemical shift mapping method described for
the RhoGDI-Rac1 complex.25 Figure 4(a) shows a
comparison of the chemical shift mapping of Rac1
interactions with the two GDIs. The most signi®cant feature in the data is that the chemical shifts
of the amide resonances of residues 7 to 18 of
RhoGDI, but not of D4GDI, are affected on formation of the respective complex, indicating that
Rac1 binds D4GDI somewhat differently from
RhoGDI, in terms either of the dynamics or of the
conformation of this N-terminal region.
RhoGDI and D4GDI have equal activity in
inhibition of GDP dissociation from Rac1
and RhoA
While both RhoGDI and D4GDI have been previously shown to inhibit GDP dissociation from
Rho family GTPases, we have used puri®ed recom-
Figure 3. Two-dimensional TROSY spectra of N-terminal domains of 2H,15N-labelled RhoGDI (left panel) and
D4GDI (right panel) in the free form (black) and with the addition of unlabelled Rac1 (red). Resonance assignments
are indicated on the spectra. The threshold of the spectra is chosen to show only the most intense signals originating
from the N-terminal ¯exible domains, and a few signals from short ¯exible C termini (labelled in italics).
127
Structure and Activity of Flexible Protein Domains
Figure 4. (a) Chemical shift mapping of interactions between Rac1
and the N-terminal domains of
RhoGDI and D4GDI. Residues of
the GDI whose amide resonances
are signi®cantly affected by Rac1
binding
(see
Materials
and
Methods) are marked red. (b) A
scheme showing the location of the
regions that are important for the
inhibition of GTP hydrolysis and
GDP dissociation. The shading of
the cylinders (corresponding to the
transient helices in RhoGDI and
D4GDI, identi®ed in the current
work) re¯ects the relative persistence of the helices. The sites of
in vivo50 and in vitro23 (unpublished
work) proteolysis are also indicated.
binant proteins in order to compare their activities
directly. Sf9 cell-expressed isoprenylated Rac1 and
RhoA were preloaded with [3H]GDP, and the ability of RhoGDI and D4GDI to inhibit dissociation of
the nucleotide was determined. The dissociation of
[3H]GDP from Rac1 and RhoA was totally blocked
by both RhoGDI and D4GDI at a molar ratio of
one (GTPase) to four (GDI) (Figure 5). Measurements at various concentrations of RhoGDI and
D4GDI showed that the two GDIs have essentially
equal activity in the inhibition of GDP dissociation
from Rac1, with maximum activity at a molar ratio
of nearly one to one (data not shown). Previous
studies with GST-fusion GDIs showed that D4GDI
is 10-20-fold less effective as a GDP-dissociation
inhibitor towards isoprenylated Cdc42Hs41 and
that the af®nity of D4GDI for Cdc42Hs is 15-fold
weaker than the binding of RhoGDI to Cdc42Hs.42
The difference between these observations on Rac1
and previously reported data for Cdc42Hs is under
further investigation.
D4GDI is less effective than RhoGDI in
inhibiting GTP hydrolysis by Rac1 and RhoA
The ability of RhoGDI to interact with the GTPbound form of GTPase targets and inhibit their
ability to hydrolyze the GTP has been reported,43-45
but to our knowledge this has not been examined
with D4GDI. We compared the ability of RhoGDI
and D4GDI to inhibit [g-32P]GTP hydrolysis by
Rac1 and RhoA. It is interesting that whereas the
RhoGDI and D4GDI exhibited similar ability to
interact with the GDP-bound forms of Rac1 and
RhoA (see above), and inhibit GDP dissociation,
the ability of D4GDI to prevent [g-32P]GTP
hydrolysis by Rac1 and RhoA was substantially
less than that of RhoGDI; this was particularly evident with RhoA (Figure 6). This was consistently
observed in multiple D4GDI preparations in which
the D4GDI had the same activity as RhoGDI to
inhibit [3H]GDP dissociation. The [g-32P]GTP
hydrolysis assays were performed on Rac1 with
RhoGDI and D4GDI at various concentrations
(data not shown), and the results showed that
although D4GDI had less absolute activity in inhibition of [g-32P]GTP hydrolysis by Rac1, the concentrations necessary for RhoGDI and D4GDI to
reach their maximal inhibitory effect were essentially the same. The data presented here suggests
that D4GDI has less activity in inhibiting GTPhydrolysis than RhoGDI, assuming that D4GDI
binds equally well to both the GDP and GTP forms
of isoprenylated Rac1.42
Functional studies using truncated proteins
It is notable that, whereas the folded domains of
the two RhoGDI and D4GDI show 74 % sequence
identity, the similarity between the two GDIs varies along the N-terminal sequence, with the ®rst 25
amino acid residues showing 16 % and residues 2669 showing 66 % sequence identity (Figure 4(a)). In
order to investigate further the functional roles of
the N-terminal domain, a series of RhoGDI deletion mutants were examined. Progressive removal
of the RhoGDI N terminus resulted in rapid loss in
the ability of RhoGDI to inhibit intrinsic GTP
hydrolysis by Rac1 (Figure 7(a)). Removal of the
®rst seven and 14 amino acid residues (N7,
N14) caused a partial loss of activity, while
removal of 20 (N20) or 30 (N30) residues
caused almost complete loss of activity to inhibit
GTP hydrolysis. This was not due to a change in
af®nity for the GTPase, as there was no further
increase in inhibitory activity when the amount
of the RhoGDI mutant used in the assay was
increased from sevenfold excess over Rac1 to
14-fold or 28-fold. Essentially the same results
were obtained when we examined the effect of
these deletion mutants on p190 GAP-stimulated
GTP hydrolysis by Rac1 or RhoA. By contrast, the
128
Structure and Activity of Flexible Protein Domains
Figure 5. Inhibition of [3H]GDP dissociation by GDIs.
The inhibitory activities of both RhoGDI and D4GDI on
the dissociation of [3H]GDP from isoprenylated (a) Rac1
and (b) RhoA were determined at concentrations of
70 nM for Rac1 and RhoA, and 280 nM for RhoGDI and
D4GDI. Results shown are representative of three or
more experiments, with the estimated experimental
uncertainty of less then 5 %. Control ( & ); plus RhoGDI
(*); plus D4GDI (*).
inhibition of GDP dissociation by these truncated
proteins (N7, N14, N20, N30) was indistinguishable from the activity of the full-length
protein (Figure 7(b)). The ability to inhibit GDP
dissociation was only lost upon removal of 41
amino acids or more from the N terminus.
As noted in the Introduction, RhoGDI controls
the partitioning of the GTPase between cytosol and
membrane. We have examined the ability of fulllength and truncated versions of RhoGDI to extract
Rac1 from endogeneous membranes when GDI
and Rac1 were co-expressed (Table 1). The distribution of Rac1 between membrane and cytosol in
cells co-transfected with Rac1 and with empty
vector was compared with the distribution in cells
co-transfected with Rac1 and the different versions
of GDI. Full-length RhoGDI effectively extracted
Rac1-GDP from membranes, and was still relatively effective at extracting Rac1-GTP (RacQ61L).
Removal of the ®rst 20 amino acid residues (20)
from RhoGDI had no effect on its activity towards
Rac1-GDP, but removal of the ®rst 40 amino acid
residues (40) totally abolished it (the lower percentage of cytosolic Rac1Q61L evident in the 41
cotransfected cells is due to a decreased expression
of endogenous RhoGDI, as shown by Western blotting). In contrast, truncation of the ®rst 20 amino
Figure 6. Inhibition of [g-32P]GTP hydrolysis by GDIs.
The inhibitory effects of both RhoGDI and D4GDI on
the hydrolysis of [g-32P]GTP by (a) Rac1 and (b) RhoA
were determined at concentrations of 70 nM for Rac1
and RhoA, and 420 nM for RhoGDI and D4GDI. Results
shown are representative of three or more experiments,
with the estimated experimental uncertainty of less than
10 %. Control ( & ); RhoGDI (*); D4GDI (*).
acid residues (20) from RhoGDI totally removed
its ability to extract RacQ61L. As expected, D4GDI
(full-length) was signi®cantly less effective than
RhoGDI at extracting the GDP-bound form of
Rac1. These experiments show that the effects of
N-terminal truncation on the activity of RhoGDI
are manifested in intact cells as well as in in vitro
assays. They also show that the ®rst 20 residues of
RhoGDI are speci®cally involved in the inhibition
of the GTPase activity, and in the regulation of
membrane partitioning of Rac1-GTP, whilst not
being important for the inhibition of GDP dissociation or in the regulation of membrane partitioning of Rac1-GDP.
Peptides derived from the N terminus of
RhoGDI inhibit Rac1 function in the
NADPH oxidase
Since the N terminus of RhoGDI was found to
be important for interaction with the GTP-bound
state of Rac1 (and RhoA), we examined whether
peptides derived from this region of RhoGDI
might serve as inhibitors of Rac1-GTP function in
a biological assay, namely the cell-free NADPH
oxidase system. Formation of superoxide anion, a
Rac1-dependent process in human neutrophils46,47
Table 1. Distribution of Rac1 between membrane and cytosol, comparing cells which were co-transfected with Rac1
and empty vector with cells co-transfected with Rac1 and different versions of GDIs (see Methods)
Empty vector
Membrane
(%)
A. RAC1 WT
56
B. RAC1Q61L
60.4
RhoGDI
D4GDI
20
41
Cytosol
(%)
Membrane
(%)
Cytosol
(%)
Membrane
(%)
Cytosol
(%)
Membrane
(%)
Cytosol
(%)
Membrane
(%)
Cytosol
(%)
44
8.9
91.1
45
55
14.2
85.8
62.9
37.1
39.5
40.7
59.2
66.1
33.9
75.8
24.2
85.6
14.4
129
Structure and Activity of Flexible Protein Domains
Figure 7. Effects of deletion mutants of RhoGDI. (a)
Inhibitory activity of RhoGDI deletion mutants toward
[g-32P]GTP hydrolysis by Rac1. The concentration of
Rac1 and deletion mutants in each experiment were
70 nM and 490 nM respectively. Control ( & ); RhoGDI
(*); N7 (*); N14(~); N20 (!); N30 (). Experimental uncertainty is less than 10 %. (b) Inhibitory
activity of RhoGDI deletion mutants towards [3H]GDP
dissociation from Rac1. Concentrations for Rac1 and deletions mutants in each experiment were 70 nM and
350 nM respectively. Control ( & ); RhoGDI (*);
N14(~); N20 (!); N30 (); N41 (*). Experimental
uncertainty is less than 5 %.
was measured in the presence or absence of short
peptides which corresponded to the ®rst 20 amino
acid residues of RhoGDI. The peptide containing
residues 5 to 20 (corresponding closely to a region,
residues 9 to 20, identi®ed as having a tendency to
form a helical structure, see above) was an effective
inhibitor of Rac1 activity in the NADPH oxidase
system, providing 100 % inhibition with respect to
the control (see Materials and Methods) at a concentration of 2 mM. Peptides 1 to 16 and 13 to 20
were somewhat less effective, but were still capable
of inhibiting superoxide generation, but the peptide 7 to 14 was much less effective, and essentially
had the same weak inhibitory activity as a series of
control peptides. Since the NADPH oxidase assay
was performed in the presence of the non-hydrolyzable guanine nucleotide, GTPgS, the observed
inhibitory effects of the RhoGDI peptides are not
due to effects on nucleotide hydrolysis.
Discussion
Functional comparison between RhoGDI
and D4GDI
The functional comparison of RhoGDI and
D4GDI by in vitro studies shows that while both
proteins are able to bind to Rac1 with similar af®nities, and are equally capable of inhibiting GDP
dissociation from Rac1 and RhoA, the same is not
true for the inhibition of GTP-hydrolysis; D4GDI is
clearly less effective at inhibiting either intrinsic- or
GAP-stimulated GTP hydrolysis by Rac1 and
RhoA. In addition, in vivo co-transfection studies
revealed that D4GDI was much less effective at
extracting the GDP-bound form of Rac1 from the
membrane. Comparison of the amino acid
sequence of the two GDIs suggests that the
sequence variation in the N-terminal region may
be responsible for the functional differences
between RhoGDI and D4GDI. Deletion mutants of
RhoGDI supported this idea, since removal of the
®rst 20 amino acid residues from RhoGDI resulted
in almost complete loss of its ability to inhibit GTP
hydrolysis, and to extract Rac1 GTP from the membrane, without affecting the inhibition of GDP dissociation. Removal of the ®rst 30 amino acid
residues caused little perturbation of the ability of
RhoGDI to inhibit GDP dissociation, in agreement
with a previous study,41 whilst removal of the ®rst
41 residues completely abolished it. Truncation of
RhoGDI by 41 amino acid residues also made the
GDI ineffective for Rac1-GDP extraction from the
membrane.
We therefore hypothesized that different parts of
the ¯exible N-terminal domain of the GDIs are
important for inhibition of GTPase activity and of
GDP dissociation, and that the N-terminal domains
of RhoGDI and D4GDI might be slightly different
in structure and dynamics, hence contributing to
their different functional activities (Figure 4(b)).
The role of the N-terminal domain in binding has
now, in part, been revealed by the three recent
crystal structures of GDI-GTPase complexes,26 ± 28
which show residues 35 to 55 of both RhoGDI and
D4GDI folding into a ``helix hairpin'' (segments 35
to 39 and 46 to 55 forming the helices) and making
important contacts with the switch I and II regions
of the GDP-bound form of the GTPase. Truncation
of RhoGDI by 41 amino acid residues will therefore disrupt the structure of this segment and its
interactions with the GTPase, rendering the truncated protein ineffective at inhibiting GDP dissociation and at extracting GDP-bound GTPase
from the membrane. In addition, in the extreme
N-terminal regions, the crystal structures of Cdc42RhoGDI27 show a short helix between residues 10
to 15, while in the structure of the D4GDI-Rac2
complex,28 the positions of residues 1 to 21 could
not be inferred from the electron density map. This
difference in the extreme N-terminal regions could
explain the functional difference of the two GDIs
in their abilities to inhibit GTP hydrolysis (see
above). Further explanation for this difference
must await the structure of a GDI with the GTPbound form of a GTPase.
Presence and significance of intrinsically
unstructured regions
The detailed analysis of the conformational
properties of the N-terminal domain in RhoGDI
and D4GDI reported here provides a consistent
picture of this highly ¯exible part of these proteins.
The NMR data demonstrates that there are subtle
conformational and/or dynamic differences which
correlate with and may account for the observed
functional differences between the two proteins.
130
We have previously reported that residues in the
region 48-58 in RhoGDI have a tendency to form a
helix but exist in solution as an equilibrium
between helical and random coil conformations;25
a similar observation is made here for residues 46
to 55 of D4GDI. In both RhoGDI and D4GDI, this
helical tendency extends further towards the N terminus, up to residue 36, although the helix in residues 36 to 46 is less stable. This helical region is
followed by very ¯exible loops (residues 58 to 63
for RhoGDI and 57 to 64 for D4GDI) connecting
the N-terminal domain to the folded part of the
protein, and preceded by another loop, residues 28
to 35 (Figure 4(b)). These loop regions have been
consistently identi®ed by accessibility to a paramagnetic probe, by NOEs and by reduced spectral
density mapping. They explain the in vitro proteolytic cleavage characteristics of both proteins
(unpublished work) where positions K33, R58 in
RhoGDI and D58 in D4GDI are particularly susceptible to proteolysis (Figure 4(b)).
All the data are therefore consistent with the
proposals that residues 36 to 58 of the N-terminal
domain of RhoGDI and D4GDI exist in a equilibrium between helical and random coil conformations in the uncomplexed proteins, adopting a
helix-turn-helix structure in the complex and playing an essential role in the binding of the GDIs to
the GDP-bound form of the rho family GTPases
and in inhibiting GDP dissociation.
Further evidence for the importance of the transient helices in mediating interactions between the
GDIs and Rac1 comes from mutagenesis.25 In the
crystal structures of the GDI-GTPase complexes,27,28 leucine residues 55 and 56 are important for
stabilizing the helix hairpin structure within the
GDI, which in turn creates a hydrophobic binding
surface for the switch II region of the GTPase. A
double mutant in which the leucine residues were
replaced by serine in either RhoGDI or D4GDI led
to a drastic decrease in the af®nity of the GDI for
Rac1-mantGDP. The NMR relaxation data on the
double mutant protein provide evidence that the
transient helical structure is perturbed in the
uncomplexed mutant (unpublished results).
Mutation of these two leucine to serine residues
appears to markedly decrease the helical propensity of this essential binding segment and, as a consequence, the interactions with the GTPase.
Both GDIs have, in addition, a region within the
®rst 30 amino acid residues which shows some tendency to form a helix, though the equilibrium
between helical and random coil conformers is
clearly less in favour of the helical state than for
residues 46 to 58. It is interesting that the location
of this N-terminal helix is different in the two
GDIs: residues 9 to 20 in RhoGDI and 20 to 25 in
D4GDI (Figure 4(b)). As highlighted earlier, the
recent crystal structures of RhoGDI and D4GDI
complexed with a GTPase27,28 show that there are
possible differences in structure and conformation
of the extreme N termini of RhoGDI and D4GDI
when complexed with the GDP-form of the
Structure and Activity of Flexible Protein Domains
GTPase (see above). It is likely that the extreme Nterminal region exists in several conformations in
these complexes, and that the conformation
adopted in the crystal structure of RhoGDI-Cdc42
complex27 is only one of these, perhaps stabilized
by crystal packing forces. It is also possible that the
populations of the different conformations differ
between GDIs. The presence of a helix in region 920, and the fact that the extreme N-terminal region
of RhoGDI is dynamically more constrained than
that of D4GDI may explain the functional differences between the two GDIs. Our functional studies do indeed show that the extreme N-terminal
regions of the GDIs (residues 1 to 30), while not
required for inhibition of GDP dissociation, appear
to be important for inhibition of GTP hydrolysis,
with RhoGDI being a better inhibitor of GTP
hydrolysis than D4GDI.
The published structures of GDP-bound GDIGTPase complexes26-28 do not allow us to explain
the speci®c role of the ®rst twenty amino acid residues of RhoGDI in inhibiting of GTP hydrolysis
and the apparent differences in GTP hydrolysis
between RhoGDI and D4GDI. In all three crystal
structures, the ®rst 25 amino acid residues in the
N-terminal region of the GDI are poorly ordered. It
could be that the extreme N terminus of RhoGDI
binds differently to the GTP- and the GDP-form of
the GTPase, possibly with the extreme N-terminal
region interacting with the switch regions of the
GTPase in such a way that GTP-hydrolysis is
inhibited. This hypothesis is supported by two
observations reported here: ®rst, that removal of
®rst 20 residues of RhoGDI has a different effect
on the extraction of Rac1 from the membrane in its
GDP- and GTP-bound forms, and second, that the
peptide 5 to 20 from RhoGDI serves as an inhibitor
of Rac1-GTP function in the cell-free NADPH oxidase system, possibly due to an inhibition of the
interaction of active Rac1-GTPgS with effector proteins. The GTP-induced change in the conformation around the switch I region of the GTPase
might promote binding of the N-terminal region of
RhoGDI, since it has previously been shown that
binding of GTPgS to RhoA produced an exposed
hydrophobic patch around the switch I effector
binding region.48 The existence of a transient helix
within the ®rst 20 residues may favor the interactions between RhoGDI and the GTP-form of the
GTPase in contrast to D4GDI which does not have
a helix in the region 9 to 20 (only a weak helical
tendency in residues 20 to 25) in the free state.
What is the biological advantage of a highly ¯exible, largely unfolded structure for the N-terminal
domain of the GDIs, a domain which is clearly
essential for their function? The crystallographic
data shows that parts of this domain adopt wellde®ned conformations on binding to the GTPase,
although it retains signi®cant mobility,27,28 as also
shown by the NMR data.25 As has been discussed
elsewhere1,49 the requirement for a folding transition on binding, driven by the binding energy,
may contribute to increasing the speci®city of the
131
Structure and Activity of Flexible Protein Domains
interaction, only optimally ``correct'' interactions
will have suf®cient binding energy to overcome
the cost of the folding transition to form a high
af®nity complex. In such a case, the existence of
transient local structures of the kind described here
can substantially decrease the entropic cost of
binding.40 The correlation between the absence of a
transient helix in residues 7 to 20 of D4GDI and
the absence of a stable interaction between this
part of the protein and Rac2 in the complex is an
example of this effect. The existence of a ¯exible
domain also opens up the possibility of increased
``versatility'' in binding, recognizing different binding partners, as proposed7 for p21Waf1/Cip1, and as
observed in the current studies where RhoGDI is
found to be involved in interactions with both the
GDP and the GTP-bound forms of a GTPase. Here,
case, given the known differences between the
GDP and GTP-bound forms of the GTPases, it is
possible that the binding of RhoGDI to the GDPbound form, to inhibit GDP dissociation, may
require recognition of a somewhat different binding surface from that involved in binding to the
GTP-bound form to inhibit GTP hydrolysis.
Finally, the existence of a ¯exible domain would
be expected to make the protein more susceptible
to proteolysis, and this is clearly the case for
RhoGDI and D4GDI. It is possible that this has
regulatory signi®cance, since in haematopoetic
cells cleavage of D4GDI by IL-1b converting
enzyme in the highly ¯exible loop 57-64 leads
to inactivation of the GDI for binding to rho
GTPases.50 Further work will be required to understand the primary physiological role of the marked
mobility of the N-terminal domain of GDIs, but the
detailed structure-function-dynamics relationships
established here, and the recent crystal structures
of the complexes, provide the basic information on
which this understanding can be built.
Materials and Methods
Materials
[3H]GDP (25-50 Ci/mmol) and [g32P]GTP (6000 ci/
mmol) were purchased from Du Pont-New England
Nuclear. Mant-GDP was purchased from Molecular
Probes Inc. and hyTEMPO from Sigma Chemical Co. All
other reagents were of the highest grade commercially
available. P190GAP was a gift from Dr Jeffrey Settleman.
PCR cloning, expression and purification of
human D4GDI
The full-length human D4GDI cDNA was cloned by
PCR ampli®cation from lgt11 cDNA library constructed
from Me2SO4-differentiated HL-60 cells (GeneBank
Accession number L20665). Two oligonucleotides were
designed as primers based on the published D4GDI
sequence.16,17 The Nhe1 sequence was added to the 50 -primer and a BamH1 sequence was added to the 30 -primer
to facilitate subcloning into a bacterial expression vector,
pET-11d (Novagen). Five independent cDNA clones
were sequenced, and con®rmed that the amino acid
sequence of the D4GDI produced here was consistent
with that of Scherle et al.,17 and different from that published by Lelias et al.,16 by having an arginine residue at
position 169 and a glycine residue at position 170.
Construction of expression vectors for RhoGDI
deletion mutants
Bacterial expression vectors for a series of deletion
mutants truncated from N-terminal end of RhoGDI were
constructed by PCR ampli®cation and subcloned into a
pET-11d vector at the Nco1 and BamH1 sites. Human
RhoGDI cDNA was used as a template in the PCR
ampli®cations. The 50 -primer was designed to have an
initiation codon in front of the indicated truncation sites
of each mutant, and the 30 -primer was designed to cover
the entire 30 -end of RhoGDI.
Expression and purification of Rac1 and RhoA
Recombinant isoprenylated Rac1 and RhoA were puri®ed from cell membranes after protein expression in a
baculovirus/Sf9 insect cell system, as previously
described by Xu et al.51 Non-isoprenylated Rac1 was
expressed in the expression vector pET11a (Pharmacia)
in Escherichia coli B834, as previously described by Lian
et al.25
Expression and purification of recombinant GDIs
All the constructed vectors for D4GDI and deletion
mutants were transformed into E. coli strain BL21(DE3)
(Novagen). Expression of recombinant proteins was
induced with 1 mM IPTG for two hours in cells growing
at 37 C when the A600 reached 0.7-0.9. GDI proteins
were puri®ed by gel ®ltration chromatography as previously described by Chuang et al.44 Further puri®cation
was performed with FPLC using a Mono Q column
(Pharmacia) eluted with a 30 ml linear gradient from 00.3 M NaCl. The isolated proteins were 95-99 % pure as
estimated by Coomassie blue staining after SDS-PAGE.
Concentrations of the puri®ed proteins were determined
with a Coomassie protein assay (Pierce). The production
of isotopically labelled D4GDI for the NMR studies was
made using the same protocol as for RhoGDI, which
was described previously by Lian et al.25 For the preparation of (2H,15N)-labelled proteins, the cells were
grown in minimal media in 2H2O with the addition of
99 % [15N]NH4Cl.
In-vivo binding assay for wild-type and
mutant RhoGDI
Hela cells were cultured in DMEM supplemented
with 10 % (w/v) bovine calf serum. Cells were plated at
a density of 1.2 106 cells per 10-cm dish 24 hours prior
to transfection. Cells were also co-transfected with mycRac1wt or myc-Rac1Q61L and either RhoGDI, D4GDI,
RhoGDI lacking the ®rst 20 (20) or ®rst 41 (41) Nterminal amino acid residues using lipofectamine
(GIBCO BRL). After 24 hours co-transfection, cells were
trypsinized, palleted by centrifugation and re-suspended
in bomb buffer containing 10 mM Hepes (pH 7.3), 0.1 M
KCl, 3 mM NaCl, 3.5 mM MgCl2 and a protease inhibitor cocktail. Cells were lysed by nitrogen cavitation (500
psi for 20 minutes at 4 C); membrane and cytosol fractions were prepared by ultracentrifugation at 150,000 g
for 40 minutes. The membrane and cytosol were subjected to immunoblot analysis with myc antibody in
132
Structure and Activity of Flexible Protein Domains
order to localize Rac1. The levels of expression of the
myc-tagged Rac1 proteins and of the various GDI constructs were veri®ed, by immunoblotting, to be equal in
all conditions.
spectrometer) of the free protein to the nearest cross
peak in the spectra of the complex were measured as:
q
dH =0:03†2 ‡ …dN =0:3†2
[3H]GDP dissociation assay
Inhibitory activity of RhoGDI, D4GDI and RhoGDI
deletion mutants towards GDP dissociation from isoprenylated Rac1 and RhoA was determined by ®ltration
assay, as previously described by Chuang et al. (1993b).44
Values of this quantity of greater than 1.75 were considered signi®cant, and the corresponding residues were
considered as being involved in interactions with Rac1.
(2H,15N)-labelled RhoGDI and D4GDI were used to
improve the spectral resolution and to increase the
signal-to-noise ratio in the spectra.
GTP-hydrolysis assays
Paramagnetic relaxation measurements
The ability of RhoGDI, D4GDI and RhoGDI deletion
mutants to inhibit intrinsic and p190 GAP-stimulated
GTP hydrolysis by isoprenylated Rac1 and RhoA was
assessed as described by Chuang et al.44
The longitudinal relaxation rates RH
1 of amide protons
of the ¯exible domains of RhoGDI and D4GDI (protein
concentration 0.3 mM) were measured at 288 K,
600MHz, at different concentrations (0, 4 and 8 mM for
RhoGDI and 0, 2 and 4 mM for D4GDI) of the paramagnetic relaxation reagent 4-hydroxy-2,2,6,6-tetramethylpiperidinyl-1-oxy (hyTEMPO). The non-selective inversionrecovery version of the 2D 1H-15N TROSY31 experiments
were performed, using a strategy similar to that
reported.52,53 (2H,15N)-labelled proteins (ca 75 % deuteration) were used to improve spectral resolution, to
increase signal-to-noise ratio and to avoid dipole-dipole
cross-relaxation of the protons. The relaxation delay was
1.2 seconds, and variable delays after the ®rst inversion
180 proton pulse were 0.0005, 0.01, 0.1, 0.4, 0.8 and 2.5
seconds. The intensities of non-overlapping signals
observed in the spectra with various delays were ®tted
very well into the three-parameter exponential model.
The ®t of relaxation data and estimations of experimental
errors were done using Felix 97.2 software.
Fluorescence binding assay
The binding of non-isoprenylated Rac1 to RhoGDI
and D4GDI was determined by measuring the decrease
in the ¯uorescence of mantGDP (Molecular Probes Inc)
bound to Rac1 on addition of GTPase, as described by
Nomanbhoy & Cerione.42
Cell-free NADPH oxidase assay
The formation of superoxide anion was evaluated in
the cell-free human neutrophil system as described previously.47 Peptides used for inhibition studies were
RhoGDI (5-20), RhoGDI (1-16), RhoGDI (13-20), RhoGDI
(7-14), and two peptides from the p85 subunit of PI
3-kinase, ERQPAPALPPKPPKP and EKLKEKKLTPI.
Control was in the absence of added peptides. For each
peptide, the assay was performed in duplicate.
NMR spectroscopy
The complete assignment of the backbone HN, 13CO,
Ca, 15N, as well as 13Cb, Ha and Hb resonances of the
¯exible domain of D4GDI was made by means of the
three-dimensional HNCO, CBCANH, CBCA(CO)NH,
NOESY-HSQC, HMQC-NOESY-HSQC and TOCSYHSQC experiments for full-length 13C,15N- or 15Nlabelled D4GDI, 0.5 mM in 20 mM sodium phosphate,
100 mM NaCl, 1 mM DTT (pH 6.3) (90 % H2O, 10 %
2
H2O), using a Bruker DMX500 spectrometer at a sample
temperature of 288 K. All these experiments and data
analysis were carried out as described previously by
Lian et al.25 The residual secondary structure was
assessed using sequential NOE connectivities (based on
cross-peak intensities in 3D NOESY-HSQC and HMQCNOESY-HSQC spectra recorded with a mixing time of
150 ms) and CSI using the program CSI.29 The CSI
values were calculated as a ``consensus'' combination of
1 a 13 a 13 b
H , C , C and 13CO CSIs. The theoretical prediction
of helical content at residue level was made using the
program AGADIR,30 using a cutoff of 1 % to identify
regions having tendency to form helical structure.
The identi®cation of RhoGDI and D4GDI residues
involved in interactions with Rac1 (at 288 K) was based
on minimum chemical shift change mapping under the
same conditions as described previously by Lian et al.25
In brief, the distance in terms of 1H and 15N chemical
shifts dH and dN between each cross-peak in the 2D
1
H-15N TROSY31 spectra (acquired on a Bruker DRX600
13
Heteronuclear relaxation measurements
15
N R1, R2 and 15N{1H}-NOE experiments were carried
out on a Bruker DMX500 spectrometer at 288 K using
conventional techniques with incorporation of gradient
selection and sensitivity improvement.54,55 (15N,2H)labelled RhoGDI and D4GDI were used for these
measurements, to improve spectral resolution, to
increase signal-to-noise ratio and to avoid the systematic
errors in measured relaxation rates due to dipole-dipole
cross-relaxation of the protons. The R1 data were collected with variable delays of 0.005, 0.025, 0.065, 0.295,
0.6 and 1.5 seconds. The R2 data were collected with
variable delays of 7.4, 22.2, 37.0, 51.8, 81.4, 125.9, 155.5,
377.6 and 599.4 ms. Relaxation delays were 1.5 and
1.8 seconds in R1 and R2 experiments, respectively. To
evaluate 15N{1H}-NOEs, the spectra were recorded with
and without NOE enhancement. In the ®rst experiment
1
H saturation was achieved with a 3.5 seconds decoupling pulse, de®ned by a series of 120 proton pulses; in
the reference spectrum acquired without saturation this
decoupling period was replaced by a delay of the same
duration.
Analysis of relaxation data
Two-dimensional spectra were processed using
Felix97.2 software, with square-sine bell function
shifted by 50-60 ; the typical size of the transformed
matrix was 2048 points in the 1H dimension and 1024
points in the 15N dimension. Non-overlapping peak
intensities extracted from relaxation spectra were ®tted
by monoexponential equations and analysed using
133
Structure and Activity of Flexible Protein Domains
Felix 97.2 software. Errors of the relaxation parameters
were estimated based on the root-mean-square of the
noise, also using standard Felix 97.2 procedures. The
dependence of the longitudinal relaxation rate of
amide protons on the hyTEMPO concentration for
each residue of ¯exible domains were ®tted by the
equation:
0
RH
1 ˆ R1 ‡ RTEMPO ‰TEMPOŠ
where [TEMPO] is the concentration of hyTEMPO,
and RH
1 is the experimentally measured longitudinal
relaxation rate of the amide proton. The dependencies
were linear within the range of concentrations used
for most residues. The slope, RTEMPO, of these plots
for each residue was calculated using a least squares
®t procedure and was used as parameter characterizing the exposure of amide proton to the paramagnetic
probe.
The 15N relaxation data were analysed using reduced
spectral density function mapping as described in detail
elsewhere.35 ± 37 Experimentally measured 15N-relaxation
parameters R1, R2 and NOE were used to sample the
spectral density function J(o) at three frequencies 0 MHz,
50 MHz and 500 MHz (corresponding to J(0), J(N) and
J(H), respectively) using the high frequency approximation J(oH) J(oH ‡ oN) J(oH ÿ oN), in accordance
with the formulae:
3
1
3
J…0† ˆ
ÿ R1 ‡ R2 ÿ Rnoe
1†
2…3d ‡ c†
2
5
J…N† ˆ
1
7
R1 ÿ Rnoe
3d ‡ c
5
2†
1
Rnoe
5d
3†
J…H† ˆ
where
Rnoe ˆ …NOE ÿ 1†R1
gN
gH
4†
and d ˆ g2Hg2N(h/2p)2/4r6HN, c ˆ 2o2N/3, where gH and
gN are the gyromagnetic ratios for 1H and 15N nuclei
respectively; h is the Plank's constant; rHN is the length
of HN bond; is the chemical shift anisotropy of the
amide nitrogen atom; NOE ˆ I/I0, where I and I0 are
peak intensities measured in a spectra acquired with and
without saturation, respectively. The values of constants,
Ê and ˆ -160 ppm, were:
calculated for rHN ˆ 1.02 A
d ˆ 1.298 109 (rad/s)2 and c ˆ 0.864 109 (rad/s)2. Uncertainties in the J(o) values were estimated by propagation
of errors, using an in-house protocol.
Acknowledgments
This work was supported by grants from the Wellcome Trust (to GCKR), and the National Institutes of
Health (grant HL48008 to GMB), and by a Biotechnology
and Biological Sciences Research Council studentship to
D.I.H. We are grateful to Mrs M. Golovanova for technical support.
References
1. Wright, P. E. & Dyson, H. J. (1999). Intrinsically
unstructured proteins: re-assessing the protein structure-function paradigm. J. Mol. Biol. 293, 321-331.
2. Weiss, M. A., Ellenberger, T., Wobbe, C. R., Lee,
J. P., Harrison, S. C. & Struhl, K. (1990). Folding
transition in the DNA-binding domain of GCN4 on
speci®c binding to DNA. Nature, 347, 575-578.
3. Cho, H. S., Liu, C. W., Damberger, F. F., Pelton,
J. G., Nelson, H. C. & Wemmer, D. E. (1996). Yeast
heat shock transcription factor N-terminal activation
domains are unstructured as probed by heteronuclear NMR spectroscopy. Protein Sci. 5, 262-269.
4. Parker, D., Rivera, M., Zor, T., Henrion-Caude, A.,
Radhakrishnan, I. & Kumar, A., et al. (1999). Role of
secondary structure in discrimination between constitutive and inducible activators. Mol. Cell Biol. 19,
5601-5607.
5. Hershey, P. E., McWhirter, S. M., Gross, J. D.,
Wagner, G., Alber, T. & Sachs, A. B. (1999). The
Cap-binding protein eIF4E promotes folding of a
functional domain of yeast translation initiation
factor eIF4G1. J. Biol. Chem. 274, 21297-21304.
6. Fiebig, K. M., Rice, L. M., Pollock, E. & Brunger,
A. T. (1999). Folding intermediates of SNARE
complex assembly. Nature Struct. Biol. 6, 117-123.
7. Kriwacki, R. W., Hengst, L., Tennant, L., Reed, S. I.
& Wright, P. E. (1996). Structural studies of
p21Waf1/Cip1/Sdi1 in the free and Cdk2-bound
state: conformational disorder mediates binding
diversity. Proc. Natl Acad. Sci. USA, 93, 11504-11509.
8. Penkett, C. J., Red®eld, C., Dodd, I., Hubbard, J.,
McBay, D. L. & Mossakowska, D. E., et al. (1997).
NMR analysis of main-chain conformational preferences in an unfolded ®bronectin-binding protein.
J. Mol. Biol. 274, 152-159.
9. Dyson, H. J. & Wright, P. E. (1998). Equilibrium
NMR studies of unfolded and partially folded
proteins. Nature Struct. Biol. 5, 499-503.
10. WuÈthrich, K. (1998). The second decade - into the
third millennium. Nature Struct. Biol. 5, 492-495.
11. Ridley, A. J. (1996). Rho: theme and variations. Curr.
Biol. 6, 1256-1264.
12. Boguski, M. S. & McCormick, F. (1993). Proteins regulating ras and its relatives. Nature, 366, 643-654.
13. Mackay, D. J. & Hall, A. (1998). Rho GTPases. J. Biol.
Chem. 273, 20685-20688.
14. Fukumoto, Y., Kaibuchi, K., Hori, Y., Fujioka, H.,
Araki, S., Ueda, T., Kikuchi, A. & Takai, Y. (1990).
Molecular cloning and characterization of a novel
type of regulatory protein (GDI) for the rho
proteins, ras p21-like small GTP-binding proteins.
Oncogene, 5, 1321-1328.
15. Ueda, T., Kikuchi, A., Ohga, N., Yamamoto, J. &
Takai, Y. (1990). Puri®cation and characterization
from bovine brain cytosol of a novel regulatory protein inhibiting the dissociation of GDP from and
the subsequent binding of GTP to rhoB p20, a ras
p21-like GTP-binding protein. J. Biol. Chem. 265,
9373-9380.
16. Lelias, J. M., Adra, C. N., Wulf, G. M., Guillemot,
J. C., Khagad, M., Caput, D. & Lim, B. (1993). cDNA
cloning of a human mRNA preferentially expressed
in hematopoietic cells and with homology to a GDPdissociation inhibitor for the rho GTP-binding
proteins. Proc. Natl Acad. Sci. USA, 90, 1479-1483.
17. Scherle, P., Behrens, T. & Staudt, L. M. (1993). LyGDI, a GDP-dissociation inhibitor of the rhoA GTP
134
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
binding protein, is expressed preferentially in lymphocytes. Proc. Natl Acad. Sci. USA, 90, 7568-7572.
Zalcman, G., Closson, V., Camonis, J., HonoreÂ, N.,
Rousseau-Merck, M.-F., Tavitian, A. & Olofsson,
B. (1996). RhoGDI-3 is a new GDP dissociation
inhibitor (GDI) - Identi®cation of a non-cytosolic
GDI protein interacting with the small GTP-binding
proteins rhoB and rhoG. J. Biol. Chem. 271, 3036630374.
Takahashi, K., Sasaki, T., Mammoto, A., Takaishi,
K., Kameyama, T., Tsukita, S. & Takai, Y. (1997).
Direct interaction of the Rho GDP dissociation
inhibitor with ezrin/radixin/moesin initiates the
activation of the Rho small G protein. J. Biol. Chem.
272, 23371-23375.
Tolias, K. F., Couvillon, A. D., Cantley, L. C. &
Carpenter, C. L. (1998). Characterization of a Rac-1
and RhoGDI-associated lipid kinase signaling complex. Mol. Cel. Biol. 18, 762-770.
Chuang, T. H., Bohl, B. P. & Bokoch, G. M. (1993).
Biologically active lipids are regulators of Rac-GDI
complexation. J. Biol. Chem. 268, 26206-26211.
Takai, Y., Sasaki, T., Tanaka, K. & Nakanishi, H.
(1995). Rho as a regulator of the cytoskeleton. Trends
Biochem. Sci. 20, 227-231.
Keep, N. H., Barnes, M., Barsukov, I., Badii, R.,
Lian, L. Y., Segal, A. W., Moody, P. C. & Roberts,
G. C. K. (1997). A modulator of rho family G
proteins, rhoGDI, binds these G-proteins via an
immunoglobulin-like domain and a ¯exible N-terminal arm. Structure, 5, 623-633.
Gosser, Y. Q., Nomanbhoy, T. K., Aghazadeh, B.,
Manor, D., Combs, C., Cerione, R. A. & Rosen, M. K.
(1997). C-terminal binding domain of Rho GDP-dissociation inhibitor directs N-terminal inhibitory peptide to GTPases. Nature, 387, 814-819.
Lian, L. Y., Barsukov, I. L., Golovanov, A. P.,
Hawkins, D. I., Badii, R. & Sze, K. H., et al. (2000).
Mapping the binding site for the GTP-binding protein Rac-1 on its inhibitor RhoGDI-1. Struct. Fold.
Des. 8, 47-55.
Longenecker, K., Read, P., Derewanda, U., Dauter,
Z., Liu, X. & Garrard, S., et al. (1999). How RhoGDI
binds Rho. Acta Crystallog. sect. D, 55, 1503-1515.
Hoffman, G. R., Nassar, N. & Cerione, R. A. (2000).
Structure of the Rho family GTP-binding protein
Cdc42 in complex with the multifunctional regulator
RhoGDI. Cell, 100, 345-356.
Scheffzek, K., Stephan, I., Jensen, O. N., Illenberger,
D. & Gierschik, P. (2000). The Rac-RhoGDI complex
and the structural basis for the regulation of Rho
proteins by RhoGDI. Nature Struct. Biol. 7, 122-126.
Wishart, D. S. & Sykes, B. D. (1994). The 13C-shift
index: a simple method for the identi®cation of protein secondary structure using 13C chemical-shift
data. J. Biomol. NMR, 4, 171-180.
MunÄoz, V. & Serrano, L. (1994). Elucidating the folding problem of helical peptides using empirical
parameters. Nature Struct. Biol. 1, 399-409.
Pervushin, K., Riek, R., Wider, G. & WuÈthrich, K.
(1997). Attenuated T2 relaxation by mutual cancellation of dipole-dipole coupling and chemical shift
anisotropy indicates an avenue to NMR structures
of very large biological macromolecules in solution.
Proc. Natl Acad. Sci. USA, 94, 12366-12371.
Mal, T. K., Matthews, S. J., Kovacs, H., Campbell,
I. D. & Boyd, J. (1998). Some NMR experiments and
a structure determination employing a {15N,2H}
enriched protein. J. Biomol. NMR, 12, 259-276.
Structure and Activity of Flexible Protein Domains
33. Kay, L. E. (1998). Protein dynamics from NMR.
Nature Struct. Biol. 5, 513-517.
34. Lipari, G. & Szabo, A. (1982). Model-free approach
to the interpretation of nuclear magnetic resonance
relaxation in macromolecules. 1. Theory and range
of validity. J. Am. Chem. Soc. 104, 4546-4559.
35. Peng, J. W. & Wagner, G. (1992). Mapping of spectral-density functions using heteronuclear NMR
relaxation measurements. J. Magn. Reson. 98, 308332.
36. Farrow, N. A., Zhang, O., Forman-Kay, J. D. & Kay,
L. E. (1995). Comparison of the backbone dynamics
of a folded and an unfolded SH3 domain existing in
equilibrium in aqueous buffer. Biochemistry, 34, 868878.
37. Farrow, N. A., Zhang, O., Szabo, A., Torchia, D. A.
& Kay, L. E. (1995). Spectral density function mapping using 15N relaxation data exclusively. J. Biomol.
NMR, 6, 153-162.
38. Dayie, K. T., Wagner, G. & Lefevre, J.-F. (1996). Theory and practice of nuclear spin relaxation in proteins. Annu. Rev. Phys. Chem. 47, 243-282.
39. Hyre, D. E. & Klevit, R. E. (1998). A disorder-toorder transition coupled to DNA binding in the
essential zinc-®nger DNA-binding domain of yeast
ADR1. J. Mol. Biol. 279, 929-943.
40. Bracken, C., Carr, P. A., Cavanagh, J. & Palmer,
A. G., III (1999). Temperature dependence of intramolecular dynamics of the basic leucine zipper of
GCN4: implications for the entropy of association
with DNA. J. Mol. Biol. 285, 2133-2146.
41. Platko, J. V., Leonard, D. A., Adra, C. N., Shaw,
R. J., Cerione, R. A. & Lim, B. (1995). A single residue can modify target-binding af®nity and activity
of the functional domain of the rho-subfamily GDP
dissociation inhibitors. Proc. Natl Acad. Sci. USA, 92,
2974-2978.
42. Nomanbhoy, T. K. & Cerione, R. (1996). Characterization of the interaction between rhoGDI and
Cdc42Hs using ¯uorescence spectroscopy. J. Biol.
Chem. 271, 10004-10009.
43. Hart, M. J., Maru, Y., Leonard, D., Witte, O., Evans,
T. & Cerione, R. A. (1992). A GDP dissociation
inhibitor that serves as a GTPase inhibitor for the
Ras-like protein Cdc42Hs. Science, 258, 812-815.
44. Chuang, T. H., Xu, X., Knaus, U. G., Hart, M. J. &
Bokoch, G. M. (1993). GDP dissociation inhibitor
prevents intrinsic and GTPase activating proteinstimulated GTP hydrolysis by the rac GTP-binding
protein. J. Biol. Chem. 268, 775-778.
45. Hancock, J. F. & Hall, A. (1993). A novel role for
rhoGDI as an inhibitor of GAP proteins. EMBO J.
12, 1915-1921.
46. Bokoch, G. M. (1995). Regulation of the phagocyte
respiratory burst by small GTP-binding proteins.
Trends Cell Biol. 5, 109-113.
47. Heyworth, P. G., Knaus, U. G., Xu, X., Uhlinger,
D. J., Conroy, L., Bokoch, G. M. & Curnutte, J. T.
(1993). Requirement for posttranslational processing
of Rac GTP-binding proteins for activation of
human neutrophil NADPH Oxidase. Mol. Biol. Cell,
4, 261-269.
48. Ihara, K., Muraguchi, S., Kato, M., Shimizu, T.,
Shirakawa, M. & Kuroda, S., et al. (1998). Crystal
structure of human RhoA in a dominantly active
form complexed with a GTP analogue. J. Biol. Chem.
273, 9656-9666.
Structure and Activity of Flexible Protein Domains
49. Spolar, R. S. & Record, M. T. (1994). Coupling of
local folding to site-speci®c binding of proteins to
DNA. Science, 263, 777-784.
50. Na, S., Chuang, T. H., Cunningham, A., Turi, T. G.,
Hanke, J. H., Bokoch, G. M. & Danley, D. E. (1996).
D4-GDI, a substrate of CPP32, is proteolyzed during
Fas-induced apoptosis. J. Biol. Chem. 271, 1120911213.
51. Xu, X., Barry, D. C., Settleman, J., Schwartz, M. A.
& Bokoch, G. M. (1994). Differing structural requirements for GTPase-activating protein responsiveness
and NADPH oxidase activation by Rac. J. Biol.
Chem. 269, 23569-23574.
52. Petros, A. M., Neri, P. & Fesik, S. W. (1992). Identi®cation of solvent-exposed regions of an FK-506 analog, ascomycin, bound to FKBP using a
paramagnetic probe. J. Biomol. NMR, 2, 11-18.
135
53. Gillespie, J. R. & Shortle, D. (1997). Characterization
of long-range structure in the denaturated state of
staphylococcal nuclease. I. Paramagnetic relaxation
enhancement by nitroxide spin labels. J. Mol. Biol.
268, 158-169.
54. Kay, L. E., Nicholson, L. K., Delaglio, F., Bax, A.
& Torchia, D. A. (1992). Pulse sequences for
removal of the effects of cross-correlation between
dipolar and chemical shift anisotropy relaxation
mechanism on the measurement of heteronuclear
T1 and T2 values in proteins. J. Magn. Reson. 97, 359375.
55. Kay, L. E., Keifer, P. & Saarinen, T. (1992).
Pure absorption gradient enhanced heteronuclear
single quantum correlation spectroscopy with
improved sensitivity. J. Am. Chem. Soc. 114, 1066310665.
Edited by P. E. Wright
(Received 10 July 2000; received in revised form 19 October 2000; accepted 19 October 2000)