(Received for publication, October 28, 1996, and in revised form, May 1, 1997)
From the ¶ Department of Biochemistry, Emory University
Medical School, Atlanta, Georgia 30322, the
Department of Biochemistry, Aichi Medical University,
Nagakute Aichi 480-11, Japan, and the ** National Institute for Medical
Research, Mill Hill, London NW7 1AA, United Kingdom
Activation of the respiratory burst oxidase
involves the assembly of the membrane-associated flavocytochrome
b558 with the cytosolic components
p47phox, p67phox, and the small GTPase Rac. Herein, the
interaction between Rac and p67phox is explored using
functional and physical methods. Mutually facilitated binding
(EC50) of Rac1 and p67phox within the NADPH oxidase
complex was demonstrated using steady state kinetic methods measuring
NADPH-dependent superoxide generation. Direct binding of
Rac1 and Rac2 to p67phox was shown using a fluorescent analog
of GTP (methylanthraniloyl guanosine-5-[
,
-imido]triphosphate)
bound to Rac as a reporter group. An increase in the methylanthraniloyl
fluorescence was seen with added p67phox but not
p47phox, and the emission maximum shifted from 445 to 440 nm.
Rac1 and Rac2 bound to p67phox with a 1:1 stoichiometry and
with Kd values of 120 and 60 nM,
respectively. Mutational studies (Freeman, J., Kreck, M., Uhlinger, D. J., and Lambeth, J. D. (1994) Biochemistry 33, 13431-13435; Freeman, J. L., Abo, A., and Lambeth, J. D. (1996)
J. Biol. Chem. 271, 19794-19801) previously
identified two regions in Rac1 that are important for activity: the
"effector region" (residues 26-45) and the "insert region"
(residues 124-135). Proteins mutated in the effector region
(Rac1(N26H), Rac1(I33N), and Rac1(D38N)) showed a marked increase in
both the Kd and the EC50, indicating that mutations in this region affect activity by inhibiting Rac binding
to p67phox. Insert region mutations (Rac1(K132E) and L134R),
while showing markedly elevated EC50 values, bound with
normal affinity to p67phox. The structure of Rac1 determined by
x-ray crystallography reveals that the effector region and the insert
region are located in defined sectors on the surface of Rac1. A model
is discussed in which the Rac1 effector region binds to
p67phox, the C terminus binds to the membrane, and the insert
region interacts with a different protein component, possibly
cytochrome b558.
Neutrophils and macrophages reduce molecular oxygen with NADPH to
produce superoxide (O2) and secondarily derived reactive oxygen species (H2O2, HOCl, OH·), which
function to kill phagocytosed microorganisms (1-4). Superoxide
generation is catalyzed by an NADPH oxidase (also called the
respiratory burst oxidase), which is dormant in resting cells but
becomes active upon exposure to bacteria or to a variety of soluble
stimuli. The enzyme consists of both cytosolic and plasma
membrane-associated protein factors. Flavocytochrome
b558 is a membrane-associated heterodimer (5-7) that contains putative binding sites for NADPH, FAD, and heme (8-11)
and therefore represents the enzymatic component of the NADPH oxidase.
Three cytosolic components (p47phox, p67phox, and a
small molecular weight GTP-binding protein, Rac), activate superoxide
generation and can be considered to be regulatory subunits of the
flavocytochrome. p47phox, while not essential for activity,
functions as a regulated adaptor protein that increases the binding of
p67phox to the oxidase 100-fold (12). p47phox appears
to act by coupling p67phox to the 22-kDa subunit of the
flavocytochrome b558 (13-15). p47phox
and p67phox exist in the cytosol of resting cells as a complex
along with a third component, p40phox (16, 17), which may
function as an inhibitory protein. p47phox and p67phox
translocate to the plasma membrane (18-21), and this correlates with
cell activation. In a cell-free system, p47phox and
p67phox form a 1:1:1 complex with flavocytochrome
b558 (22).
The small molecular weight GTP-binding protein, Rac, occurs as two isoforms (Rac1 and Rac2) that are 92% identical in amino acid sequence (23). The two isoforms differ primarily in their C termini; Rac1 but not Rac2 contains a polybasic C terminus. In resting cells, Rac is located in a cytosolic complex with an inhibitory protein, RhoGDI (24-27). Upon cell activation, Rac2, the more abundant isoform in neutrophils (28-30), becomes associated with the plasma membrane (28), and translocation correlates with NADPH oxidase activity (28, 30, 31).1 Translocation of Rac requires neither p47phox nor p67phox and occurs with different kinetics than these other cytosolic components (34-37), suggesting that the binding of Rac to the plasma membrane is regulated by mechanisms that are distinct from those that regulate p47phox/p67phox assembly. In their isoprenylated forms, both Rac1 and Rac2 can activate superoxide generation in a cell-free system (38-40). Kinetic characterization and binding studies are complicated by the presence of the isoprenyl group, which limits the solubility and requires the presence of a detergent. The proteins in their nonisoprenylated forms can be expressed in and purified from bacteria. However, in their nonisoprenylated forms, only Rac1 activates efficiently. This is because membrane association is essential for optimal function of Rac, and nonisoprenylated Rac1 can interacts with the membrane via its polybasic C terminus (41). Bacterially expressed versions of Rac1 have therefore been used for most of the studies described herein.
We have previously characterized two regions on Rac1 that are important for its ability to activate the NADPH oxidase. These are the effector region (within the range of residues 26-45) and the insert region (residues 124-135). The effector region shows homology to a region on Ras that has been characterized as mediating the GTP-dependent binding to the Ras effector Raf-1, a member of the MAP kinase cascade (42, 43). The effector region of Ras includes residues the conformation of which changes significantly depending on whether GTP or GDP is bound. The insert region on Rac has no counterpart in Ras. Mutation of residues in both the effector region and the insert region of Rac1 results in a marked decrease in the ability to support cell-free superoxide generation (44-46), and the primary effect was decreased affinity of Rac1, based upon an increase in the EC50 (44, 47).
Rac1 has been shown to bind to immobilized p67phox (48, 49), and the interaction has been demonstrated using yeast two-hybrid analysis, but it is not clear whether Rac also interacts with other targets. In addition, no quantitative information on Rac binding to p67phox is available (e.g. affinity, stoichiometry). Herein, steady state kinetic analysis was used to demonstrate the functional linkage between Rac and p67phox. In addition, Rac binding to p67phox was measured directly, making use of a fluorescent GTP analog that binds tightly to Rac as a reporter group. The Rac-associated analog undergoes an increase in fluorescence upon interaction of Rac with p67phox, and this fluorescence change was used to quantify the Rac binding to p67phox. This method, used in conjunction with mutational analysis, reveals that the effector region participates in binding to p67phox, while the insert region does not. Mapping of residues the mutation of which lowers activity onto the structure of Rac1 (recently determined by x-ray crystallography; Ref. 50) reveals that the effector region and the insert region are located in distinct sectors of the protein, consistent with a model in which these two regions bind to distinct targets within the NADPH oxidase complex.
Cytochrome c (type VI), NADPH,
n-octyl glucoside, diisopropyl fluorophosphate,
phenylmethylsulfonyl fluoride, and
N--tosyl-L-lysine chloromethylketone were
from Sigma. GTP
S was purchased from Boehringer Mannheim. HESPAN (6%
hetastarch in 0.9% NaCl) was from American Hospital Supply Corp., and
lymphocyte separation medium (6.2% Ficoll, 9.4% sodium diatrizoate)
was obtained from Organon Tekniker. Superoxide dismutase and
dithiothreitol were from Wako Pure Chemical Co. Heparin-Sepharose
CL-6B, DEAE-Sepharose CL-6B, CM-Sepharose CL-6B,
-aminooctyl-agarose, and glutathione-Sepharose were purchased from
Pharmacia LKB. L-
-phosphatidylcholine (bovine brain),
L-
-phosphatidylethanolamine (bovine brain),
L-
-phosphatidylinositol (bovine brain), and
sphingomyelin (bovine erythrocyte) were from Sigma. All other reagents
were of the highest grade available commercially. Methyl isotoic
anhydride was purchased from Molecular Probes (Eugene, OR), and
mant-GppNHp2 was synthesized as described
previously (51).
Recombinant p67phox was expressed and purified from baculovirus-infected Hi5 insect cells as described (22, 31). Wild type and mutant Rac1 were expressed in Escherichia coli as the glutathione S-transferase fusion proteins, purified using glutathione-Sepharose beads, and cleaved from the glutathione S-transferase domain with thrombin (52). All recombinant proteins were purified to greater than 95% homogeneity.
Isolation of Human Neutrophils and Plasma Membrane PreparationHuman neutrophils were obtained from peripheral blood of normal healthy donors after obtaining informed consent. Erythrocytes were sedimented with HESPAN, and the mononuclear cells were removed from the resulting supernatant by centrifugation through lymphocyte separation medium (53). The resulting cells were greater than 95% neutrophilic granulocytes. Neutrophils were resuspended in cavitation buffer (25 mM HEPES, pH 7.4, containing 100 mM KCl, 3 mM NaCl, 5 mM MgCl2, 6 µM diisopropyl fluorophosphate, 0.5 mM phenylmethylsulfonyl fluoride, 2 µM each leupeptin, pepstatin, and aprotinin). Cells (6 × 109) in 20 ml of ice-cold buffer were disrupted by nitrogen cavitation after being pressurized at 500 p.s.i. for 20 min at 4 °C, and plasma membranes were prepared as described (54).
Purification and Reconstitution of Flavin-depleted Cytochrome b558 with FADPlasma membrane was solubilized in the
presence of 40 mM octyl glucoside and 0.5% sodium cholate
(11). Detergent-solubilized cytochrome b558 was
purified as described previously (9) with some modifications (11).
Purified FAD-depleted cytochrome b558 (15.6 nmol
of heme/mg of protein) was incubated in 50 mM Tris acetate
buffer, pH 7.45, containing 5 mM KCl, 10% glycerol, 1 mM dithiothreitol, 1 mM EGTA, 1 mM
phenylmethylsulfonyl fluoride, 2 µM each leupeptin,
pepstatin, and aprotinin (buffer B), and phospholipids
(L--phosphatidylcholine/L-
-phosphatidylethanolamine/L-
-phosphatidylinositol/sphingomyelin/cholesterol = 4:2:1:3:3 (w/w/w/w/w); lipid/protein = 100, w/w) were added along with a 10-fold excess of FAD over heme. After incubating at
4 °C for 2 h, the mixture was dialyzed against two changes of
buffer B to remove free FAD. The FAD-reconstituted material typically
contained a FAD:heme ratio of 0.4-0.5 (11).
The heme content of cytochrome
b558 reconstituted with FAD and phospholipids
was determined by reduced minus oxidized difference spectroscopy at
424-440 nm using an extinction coefficient of 161 mM1·cm
1 (55). The flavin
content of FAD-reconstituted cytochrome b558 was
estimated by the fluorimetric method (11). Fluorescence spectra were
recorded with a Hitachi model F-3000 spectrofluorimeter, and routine
fluorescence measurements were made with a Perkin-Elmer LS-5B
spectrofluorimeter. Samples (mant-GppNHp, Rac 1, cytochrome b558, and cytosolic factors) were incubated at
20 °C in 0.3 ml of 20 mM Tris acetate buffer, pH 7.45, containing 3 mM NaCl, 50 mM KCl, and 0.1 µM MgCl2. Preloading of Racs with mant-GppNHp was carried out for 15-20 min, at which point the fluorescence change
due to the guanine nucleotide binding was stable. Low MgCl2 concentration was essential to facilitate complete guanine nucleotide exchange. Titrations were carried out by adding p67phox and
recording fluorescence readings until three successive stable readings,
at least 45 s apart, were obtained. Fluorescence changes induced
by p67phox occurred rapidly (within 1-2 min) and did not
change further with prolonged incubation. Spectral resolution was 5 nm
for both the excitation and emission paths, respectively.
Superoxide
generation was measured by superoxide dismutase-inhibitable reduction
of cytochrome c as described previously (54) using a
Thermomax kinetic microplate reader (Molecular Devices, Menlo Park,
CA). Rac, preloaded with a 5-fold molar excess of GTPS for 15 min at
25 °C in the absence of MgCl2 (52), was combined with
p47phox, p67phox, 10 nM cytochrome
b558, and 1 µM FAD followed by activation
with 40 µM arachidonate in 50 mM NaCl, 4 mM MgCl2, 1.25 mM EGTA, 20 mM Tris-HCl, pH 7.0, as described (56). The mixture was
incubated at 25 °C for 5 min followed by the addition of 200 µM NADPH and 200 µM cytochrome
c. An extinction coefficient at 550 nm of 21 mM
1 cm
1 was used to calculate
the quantity of cytochrome c reduced (57).
The theoretical lines through the data shown in Figs. 1 and 2 were calculated using a nonlinear least squares fit of the data using the Michaelis-Menten equation and were plotted using Sigma Plot. Kinetic constants are reported as Vmax and EC50 (effective concentration at 50% of Vmax). Fluorescence titrations were fit to a single site binding equation as described previously (58) as follows,
![]() |
(Eq. 1) |
We previously showed that a functional interaction between p67phox and p47phox, which are known to form a complex, can be demonstrated using steady state kinetics assaying superoxide generation; in the presence of the other required components, the EC50 for each protein was found to vary inversely with the concentration of the partner protein (59). This result indicates a thermodynamic linkage between the binding of the two proteins within the NADPH oxidase complex and is consistent with a complex between the partner proteins. This approach was tried with p67phox and Rac1 under conventional assay conditions (in the presence of p47phox), but effects of varying the concentration of one protein on the EC50 of the other protein were not seen. We attribute this to the already high affinity of both components in the oxidase complex, which makes it difficult to observe further decreases in their EC50 values, and to the presence of multiple binding interactions for each of the proteins, which may obscure observation of such interactions. However, the system can be simplified, since p47phox need not be present to observe high activity when high concentrations of p67phox and Rac1 are used (12). The omission of p47phox simplifies the kinetic analysis; the 50-100-fold weaker binding of Rac1 and p67phox makes any decreases in EC50 values easy to detect, and p47phox is ruled out as a mediator of any observed kinetic linkage that must therefore be due to direct interactions among the remaining components (Rac1, p67phox, cytochrome b558). Under these conditions, the EC50 for Rac1 decreased at increasing concentrations of p67phox (Fig. 1A). Likewise, at increasing concentrations of Rac1, the EC50 for p67phox decreased (Fig. 1B). A 6-fold increase in the concentration of p67phox resulted in a roughly 5-fold decrease in the EC50 for Rac, while a 10-fold increase in Rac concentration resulted in a >2-fold decrease in the EC50 for p67phox. These data imply a functional interaction between p67phox and Rac1, consistent with a complex between these two proteins.
Characterization of the Binding of Mant-GppNHp, a Fluorescent Derivative of GTP, to Rac1We previously showed that mant-GppNHp
binding to Rac1 is accompanied by an increase in fluorescence at 445 nm, the emission peak of the mant moiety, and we have used this
increased fluorescence to quantify the binding of mant-GppNHp to Rac1
and point-mutated types of Rac1 (47). We used this fluorescence
increase in the present studies to verify that Rac1, Rac2, Rac1(N26H),
Rac1(I33N), Rac1(D38N), Rac1(M45T), Rac1(K132E), and Rac1(L134R) all
bind mant-GppNHp tightly and achieve maximal binding within the same time period. Quantifying binding as described previously, all of these
recombinant forms of Rac bound the guanine nucleotide with an
approximate 1:1 stoichiometry and with apparent Kd values in the 1-20 nM range. In addition, all had achieved
maximal fluorescence change by about 5 min, well within the 15-20 min incubation period used for preloading of Rac1 with the nucleotide. Examination of the Rac1 structure (50) shows that the side chains of
these mutants do not directly interact with the nucleotide, are
relatively exposed, and can accommodate the respective mutations without altering the overall fold of the protein. As a control, Rac(T17N), a mutation that in Ras renders this GTPase incapable of
binding guanine nucleotide, did not produce any fluorescence change
when added to mant-GppNHp. Thus, by preincubating mant-GppNHp in the
presence of a slight excess of Rac or Rac mutants, a fully associated
complex between the fluorescent nucleotide and Rac was formed. The
ability of mant-GppNHp to produce an active conformation of
representative versions of Rac1 was also investigated. Mant-GppNHp was
compared with GTPS (0.5 µM each) in their abilities to
support NADPH-dependent superoxide generation, using
purified oxidase components in the cell-free system. Using wild type
Rac1 (Fig. 2), the mant-GppNHp supported superoxide
generation to about 60% of the level seen with GTP
S. For
representative point-mutated forms (Rac1(N26H) and Rac1(D38N)) that
have a reduced ability to stimulate superoxide generation (44), the
mantGppNHp worked nearly as well as GTP
S.
Fig. 3A (solid
line) shows the emission spectrum for mant-GppNHp free in
solution. Upon addition of Rac1, there was a small increase in
fluorescence (about 5%), as was described previously (dotted
line). When p67phox was added, there was a further
increase in the mant fluorescence, and the emission maximum was
blue-shifted about 5 nm (Fig. 3A, dashed line).
This was not due to any direct effect of p67phox on the free
mant-GppNHp, since p67phox had no effect on its fluorescence in
the absence of Rac1 (Fig. 3C). In Fig. 3B,
p47phox was added to the mant-GppNHp·Rac complex. In contrast
to p67phox, there was no increase in the fluorescence intensity
upon the addition of p47phox (compare solid and
superimposed dotted lines). When both p47phox and
p67phox were added to the Rac(mant-GppNHp) complex
(dashed line), the fluorescence increase was essentially the
same as that produced by p67phox alone. Furthermore, the
addition of 0.25 mM arachidonate caused no significant
effect on the fluorescence enhancement due to the binding of
p67phox to Rac(mant-GppNHp), indicating that the guanine
nucleotide-dependent binding of Rac1 to p67phox
occurs whether or not arachidonate is present.
The increase in fluorescence of Rac(mant-GppNHp) was used to quantify
the strength of binding of Rac to p67phox. Fig.
4 shows the titration of Rac1(mant-GppNHp) with
p67phox and p47phox. As above, p47phox had no
effect on the fluorescence of Rac(mant-GppNHp), while p67phox
produced a saturable increase in the fluorescence. The increase in
fluorescence fit a theoretical curve (Fig. 4, solid line)
for single site binding of p67phox to Rac (i.e. no
cooperativity was detected). In the experiment shown, the apparent
binding constant was 170 nM, somewhat weaker than published
values for the EC50 (50 nM) for Rac in
cell-free superoxide generation assays (47). The average of 11 such
titrations gave an value of 124 ± 15 nM, as
summarized in Table I. The binding experiment was also
carried out in the presence of p47phox (see Table I, titration
not shown). p47phox did not affect the ability of Rac1 to bind
to p67phox. In addition, the binding experiment was also
carried out in the presence of arachidonate. Although this did not
appear to affect binding in a significant way, increased light
scattering prevented detailed quantitative analysis and did not permit
us to carry out binding and activity experiments under identical conditions.
|
Titrations of mant-GppNHp complexes of Rac1
and Rac2 with p67phox are shown in Fig. 5.
Binding of p67phox to Rac2(mant-GppNHp) gave a somewhat smaller
maximal fluorescence yield, but the calculated Kd
value was 2-fold lower than that using Rac1(mant-GppNHp), as summarized
in Table I. This slightly higher affinity of Rac2 is consistent with
studies using the yeast two-hybrid system to detect the interaction of
Rac1 and Rac2 with p67phox (49). While quantitation is not
possible using this method, the use of the Rac2 hybrid yielded a more
intense blue color in the two-hybrid assay, suggesting enhanced
binding. In contrast to the increased binding of Rac2 to
p67phox, the EC50 for Rac2 was markedly elevated
compared with Rac1 in the cell-free NADPH-superoxide generation assay
(Table I).
Point mutations of Rac1 were previously characterized (44) and shown to produce large changes in the EC50 for Rac1 in the cell-free NADPH-superoxide generation assay. Previous published values as well as unpublished experiments were averaged to obtain the EC50 values (Table I). The binding of these mutated Rac1 forms was quantified using mant fluorescence as above. Results are shown in Fig. 5, and averages of Kd values obtained in several independent experiments are summarized in Table I. Representative mutations in both the effector region and the insert region were investigated. While native Rac1 bound tightly to p67phox, mutations in the effector region weakened the binding. In particular, the mutations at the 33- and 38-positions showed large effects on binding, while those at the more extreme ends of the effector region (positions 26 and 45) produced smaller but reproducible effects. Thus, decreased binding to p67phox accounts for the decreased ability of effector region mutants to support superoxide generation. In contrast, mutations in the insert region of Rac1 produced large effects on the EC50 for superoxide generation in the cell-free system but showed no effect on the Kd for Rac1 binding to p67phox. Thus, Rac1 does not appear to utilize its insert region to bind to p67phox.
Finally, an additional region on Rac C-terminal to residue 143 for interaction with p67phox has been postulated, based on the inability of a Rac143Rho chimera (N-terminal 143 residues from Rac1, with the rest from RhoA) to bind to p67phox using overlay blotting methods (60). Using this chimera, we confirmed that the chimera was inactive in supporting superoxide generation and that it failed to bind significantly to p67phox (Table I).
Direct demonstration
of a stoichiometric complex between Rac1 and p67phox has proven
to be difficult, presumably due to the kinetic and or thermodynamic
lability of the complex. We previously attempted to demonstrate
interaction using gel filtration but were unable to detect a stable
complex.3 Binding of Rac isoforms to
p67phox has been demonstrated using p67phox (or a
p67phox-GST fusion protein) immobilized on glutathione beads
(46) or on a nitrocellulose filter (48, 60) and also by yeast
two-hybrid analysis (49). However, a large excess of Rac was used in
the first two cases, and the percentage of binding was low compared with that predicted for a 1:1 complex. Using both methods, effector region mutations disrupted the interactions, suggesting that the Rac-p67phox binding is relevant to regulation of the NADPH
oxidase. While informative, neither of these methods is quantitative,
and neither can be used to obtain direct measures of binding affinity
or stoichiometry. In the present studies, a fluorescent derivative of
GTP has proven to be effective for this purpose. The mant is bound
through the 3-hydroxyl group of the ribose ring. O3
is exposed to
solvent, making only a weak hydrogen bond with E31 (50). The
mant-GppNHp should bind to Rac1 without altering the basic interactions
between the nucleotide and the protein, thus serving as a good reporter for binding. The quantum yields of mant guanine nucleotides shows high
sensitivity to solvent polarity and to small changes in protein environment (51), and mant-modified nucleotides have been used to
demonstrate both binding of nucleotides to their binding proteins (47,
63, 64) and the interaction of the guanine nucleotide-protein complex
with a second protein (58). In the latter study, the complex between
RhoGDI and Cdc42Hs has been demonstrated, based on quenching of the
fluorescence of the Cdc42Hs(mant-GDP) complex upon interaction with
RhoGDI.
A requirement for the use of this method is that the fluorescent group
must bind tightly to one of the interacting proteins and that it should
induce an active conformation of the protein. The complex between
mant-GppNHp and Rac1 was therefore initially characterized. As is shown
in Fig. 3, complex formation was accompanied by a small increase in the
fluorescence of the mant moiety. As was shown previously, this
fluorescence change was used to quantify the binding of mant-GppNHp to
Rac1 (47). In agreement with earlier studies, the complexes show high
affinity, with apparent Kd values ranging from 1 to
20 nM for native and point mutated forms of Rac. Mutant
forms of Rac showing elevated Kd values for guanine
nucleotide binding (including Rac1(A59T) and Rac1(T75K)) were
eliminated from consideration in this study. The mant-GppNHp generates
an active conformation of Rac1, albeit somewhat less active than that
produced by GTPS. We presume that the binding affinities generally
reflect those that occur in the active complex with nonfluorescent
nucleotide, but it is possible that these are perturbed in subtle ways
due to the presence of the fluorescent group.
Using the fluorescence method we find that Rac(mantGppNHp) binds
to p67phox with a Kd of 110 nM.
While this is a moderately high affinity, it is considerably weaker
than the binding of many signaling complexes, perhaps accounting for
the instability of this complex to gel filtration chromatography and
the low stoichiometry of binding found using other methods. The
EC50 for Rac1 in activating superoxide generation is 60
nM (the actual EC50 is probably less, since
this represents an upper limit value; see Ref. 47). The most likely
explanation for the lower value for the EC50 is that other
binding interactions in addition to p67phox participate in Rac
binding within the NADPH oxidase complex.
Table II lists most of the point mutations that have been characterized either in our own laboratory or in those of other labs and depicts effects on activity and on binding to p67phox. Because the methodologies used and the expression of data are not directly comparable, the relative activity is indicated by addition symbols (+), with ++++ representing normal activity, and binding is summarized as either "normal" or "weak." Inspection of Table II reveals two regions, the effector region and the insert region, that have large effects on activity and reveals additional regions (e.g. residues in the range of residues 61-105) that have small or no effects. The effector region has been studied extensively by several groups, and a large number of mutations have been made. Except for conservative replacements (e.g. Rac1(I33V)), most changes in this region have large effects on the ability of Rac1 and Rac2 to activate cell-free superoxide generation. The structure of Rac1-GppNHp was recently determined by x-ray crystallography (50) and is shown in Fig. 6A, with the guanine nucleotide indicated in violet. The overall fold of the protein is very similar to that of Ras except for two regions. The insert region, which forms a well defined, exposed, helical domain, and residues 28-38, which are very flexible and are less defined in the x-ray structure (see Ref. 50 for a discussion of these two regions). Effector region mutations summarized in Table II are indicated in maroon, and insert region mutations are in green. As shown in Fig. 6B, the effector region and the insert region represent distinct regions on Rac1. Each can be envisioned as forming a surface for interaction of partner proteins within the NADPH oxidase complex. Except for position 45, which is slightly more distant, all of the effector region mutations cluster closely to form a single surface. Other possible exceptions within the effector region may include threonine 35, which coordinates the magnesium, which is involved in guanine nucleotide binding (50). Other residues within this region are exposed on the surface as part of the effector loop, and mutations of these residues are unlikely to produce a long range effect on the structure of Rac1. Representative mutations (indicated by stars in Fig. 6A) within this region were investigated for their effects on binding to p67phox. These mutations resulted in effects, some quite large, on binding to p67phox. All effector region mutations also affected the EC50 for Rac1 in the cell-free assay. Quantitatively, the magnitude of the effects on binding paralleled the effects on the EC50 for mutations at residues 33 and 38, but the effects on binding were smaller than the effects on EC50 for mutations at positions 26 and 45. This may indicate that the presence of other protein component within the NADPH oxidase complex perturbs the binding of Rac1 to p67phox, particularly at the periphery of the effector region, or that the different assay conditions perturbed the binding energies. The fact that all of the mutations produced effects on both activity and binding argues strongly that the effector region is utilized within the NADPH oxidase complex for binding to p67phox.4
|
In contrast to the effector region, representative activity-affecting mutations in the insert region (K132E and L134R in Fig. 6A) had no effect on the binding to p67phox. These residues are largely exposed on the surface of the insert region, and mutations are unlikely to produce structural perturbations outside of the effector region. In addition, the mutated residues do not directly contact the guanine nucleotide, consistent with a lack of effect on guanine nucleotide binding or hydrolysis. Thus, the most likely explanation for the effects of insert region mutations on the EC50 for Rac1 is that these residues participate in binding to another component within the NADPH oxidase complex. We have previously shown that the association of the polybasic C terminus of (nonisoprenylated) Rac1 with the membrane is essential for optimal activity and that this does not occur with nonisoprenylated Rac2 (41). The insert region lies at the opposite pole from the C terminus (Fig. 6B), making unlikely that both regions interact with the membrane. Thus, the data are most consistent with the insert region interacting with a distinct protein component of the NADPH oxidase rather than with the membrane. Since insert region mutations reduce relative activity regardless of whether p47phox is present (data not shown), we speculate that the insert region binds directly to cytochrome b558. We propose a model in which a minimum of three interaction regions on Rac are important for reconstituting cell-free NADPH oxidase activity. The C terminus anchors the Rac to the membrane, while the effector region and the insert region bind, respectively, to p67phox and to another component of the oxidase, possibly cytochrome b558.
We thank Alan Hall for providing the construct for the Rac143Rho chimera.