From the Institut de Biologie Structurale,
CEA-CNRS-UJF, UMR 5075, 41 rue Jules Horowitz, 38027 Grenoble cedex 1, France and the § Laboratoire Biochimie et Biophysique des
Systèmes Intégre's/CEA-CNRS-UJF, UMR
5092/Département de Biologie Moléculaire et Structurale/CEA
Grenoble, 17 Rue des Martyrs, 38054 Grenoble cedex 9, France
Received for publication, January 30, 2001
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ABSTRACT |
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Upon activation, the NADPH oxidase from
neutrophils produces superoxide anions in response to microbial
infection. This enzymatic complex is activated by association of
its cytosolic factors p67phox,
p47phox, and the small G protein Rac
with a membrane-associated flavocytochrome b558. Here we
report the crystal structure of the active N-terminal fragment of
p67phox at 1.8 Å resolution, as well as
functional studies of p67phox mutants. This
N-terminal region (residues 1-213) consists mainly of four TPR
(tetratricopeptide repeat) motifs in which the C terminus folds back
into a hydrophobic groove formed by the TPR domain. The structure is
very similar to that of the inactive truncated form of
p67phox bound to the small G protein Rac
previously reported, but differs by the presence of a short C-terminal
helix (residues 187-193) that might be part of the activation domain.
All p67phox mutants responsible for Chronic
Granulomatous Disease (CGD), a severe defect of NADPH oxidase function,
are localized in the N-terminal region. We investigated two CGD
mutations, G78E and A128V. Surprisingly, the A128V CGD mutant is able
to fully activate the NADPH oxidase in vitro at
25 °C. However, this point mutation represents a
temperature-sensitive defect in p67phox that
explains its phenotype at physiological temperature.
The NADPH oxidase of phagocytic cells is responsible for the
production of microbicidal superoxide anions. This enzymatic complex is
activated at the onset of phagocytosis by association of its cytosolic
factors p67phox,
p47phox, and the small G protein Rac with a
membrane-associated flavocytochrome b558 (1). Mutations in
any of these components except Rac can lead to a severe immune defect
known as Chronic Granulomatous Disease
(CGD).1 The cytosolic factors
p47phox, p40phox,
and p67phox are modular proteins comprising
a set of structural domains such as SH3 domains, proline-rich regions,
and TPR motifs (2), all of which are prone to protein-protein
interactions. Through these various domains, the cytosolic factors are
able, upon stimulation, to associate, translocate to the membrane, and
finally activate the electron transfer from NADPH to O2
through the flavocytochrome b558. Over the past few years,
serious efforts have been made to delineate the nature and the
succession of intra- and intermolecular interactions between the
cytosolic factors leading to the activated complex (3, 4). Most, if not
all, pairwise interacting protein domains have been identified, but the
sequence of the molecular rearrangements as well as the structural
modifications governing this cascade are still a matter of debate. It
is known that Rac can interact in a GTP-dependent manner
with the N-terminal region of p67phox
comprising amino acids 1-199 (5). Moreover, all known missense mutations in p67phox responsible for CGD are
localized in this N-terminal region (6). This region of
p67phox has been shown to contain four TPR
motifs (2) that were first identified as degenerate 34 amino acid
sequences present in a variety of proteins from bacteria to eukaryotes
(7, 8). One TPR motif consisting of two antiparallel In this study, we report the structure at 1.8 Å resolution of the
catalytically active N-terminal region (residues 1-213) of
p67phox that contains four TPR motifs. We
compared it to the Rac-p67phox structure as
well as to other TPR domain structures. We have also investigated the
molecular and structural basis of the defects due to some of the CGD
mutations. In particular, we report biochemical and biophysical
evidence for the instability of the A128V mutant at physiological
temperature, in agreement with our structural analyses.
Native and Selenomethionyl Protein Production--
The cDNA
for the N-terminal part of p67phox
corresponding to amino acids 1-213 was obtained by polymerase chain
reaction and cloned into pET-15b (Novagen). The protein was expressed
in Escherichia coli BL21 (DE3) and purified by affinity
chromatography in two steps: first on a Ni2+-column
equilibrated in 20 mM Hepes, pH 7.5, 250 mM
NaCl and eluted with a linear gradient of imidazole and second on an
SP-Sepharose column equilibrated in 20 mM Hepes, pH 7.5 and
eluted with a linear gradient of NaCl. The protein was then
concentrated to 5-6 mg/ml with Centricon10 (Amicon).
Seleno-L-methionine (Se-Met)-labeled protein was produced
in a similar way. The protein was expressed in E. coli B834
(DE3). A 50-ml preculture in Luria-Bertani medium was used to inoculate
2 liters of a defined medium prepared as reported before (13)
supplemented with 20 mg/liter of Se-Met. The last purification step was
done in the presence of 10 mM dithiothreitol and 1 mM EDTA to avoid oxidation of selenomethionines. High
resolution electrospray ionization mass spectrometry was consistent
with 100% selenium incorporation.
CGD Mutants of p67Nter--
Mutants G78E and A128V
of p67phox-(1-213) were constructed using
site-directed mutagenesis (Stratagene kit) on the native cDNA cloned in the pET-15b vector. The mutant A128V was expressed in E. coli BL21 (DE3) at 15 °C for 16 h instead of the
3 h at 37 °C employed for the native protein. The first steps
of the purification were the same as for the native protein, but an
additional purification step was carried out by gel filtration on a
Superdex 200 Hiload 16/60 column (Amersham Pharmacia Biotech)
equlibrated in 20 mM Hepes, pH 7.5 and 200 mM
NaCl. The protein was concentrated to 5 mg/ml with Centricon10.
Limited Proteolysis--
Native
p67phox-(1-213) and the A128V mutant at a
concentration of 0.6 mg/ml were submitted to limited proteolysis for
one hour at 25 °C by trypsin (Roche Molecular Biochemicals) at a
protease/protein ratio of 1:200 (w/w).
Circular Dichroism (CD)--
CD spectra were recorded on a Jobin
Yvon CD6 dichrograph from 190 to 260 nm with a 1-mm path length cell
and a thermostated cell holder. The protein concentrations were 0.5 mg/ml. The spectra were recorded on one sample per protein from 15 to
35 °C by increments of 5 °C. At each temperature, the sample was
incubated for 15 min.
NADPH oxidase activity--
The NADPH oxidase activating potency
was assessed in a semi-recombinant cell-free system (14) containing 3.5 µg of membrane protein, 20 pmol of recombinant
p47phox, 20-400 pmol of an N-terminal
fragment of p67phox, 2 mM
MgCl2 and 10 pmol of Rac in a final volume of 200 µl. Rac was loaded with GTP- Assay of Rac Binding--
Rac fused to GST or GST alone were
immobilized on glutathione-Sepharose beads (Amersham Pharmacia
Biotech). Rac was loaded with GTP- Crystallization--
Crystals of the native protein or the
Se-Met protein were grown at 20 °C by vapor diffusion in hanging
drops by mixing equal volumes of protein (5-6 mg/ml) and reservoir
solutions (17% polyethylene glycol monomethyl ether 2000, 100 mM sodium citrate, pH 4.5, 10% glycerol, and 10 mM dithiothreitol in the case of the Se-Met protein). The
crystals grew as thin needles (20 × 20 × 200 µm3) and belong to the trigonal space group
P31 (a = b = 67.7 Å, c = 50.2 Å) with one molecule in the
asymmetric unit. Crystals exhibit various amounts of merohedral
twinning; however, non-twinned crystals could be selected after
analyzing the diffraction
data.2
Data Collection and Processing--
A native data set was
collected (beamline ID14-EH1, ESRF-Grenoble) to 1.8 Å resolution
(Table I), integrated with DENZO (15) and
scaled with SCALA (16). A SAD data set was collected on a
selenomethionyl-substituted crystal at the K absorption edge of
selenium (beamline ID29, ESRF-Grenoble). Data were integrated and
scaled with the DENZO/SCALEPACK programs. The program Shake and Bake
(17) located 5 of 7 selenium atoms expected in the asymmetric unit.
Structure Determination and Refinement--
Initial phases
calculated with MLPHARE (16) using the 5 selenium sites allowed the
location of a sixth selenium atom detected in an anomalous Fourier
difference map. Final phases from MLPHARE resulted in a figure of merit
of 0.37-2.8 Å resolution. Phases were extended to 1.8 Å on the
native data set using the program DM (16) assuming a solvent content of
45% of the unit cell volume (Table I). 85% of the model was built
automatically using the program wARP (18). Loop residues 151-158,
residues 2-3 at the N terminus and 191-193 at the C terminus were
added manually using the program O (19). The structure was refined with
CNS (20) to a final crystallographic Rfactor of
18.2% and an Rfree of 20.5% at 1.8 Å resolution (Table II). The model consists
of residues 2-193 of p67phox, 160 water
molecules, and one citrate anion, the citrate being essential for
crystallization. All non-glycine residues are in the most favored or
additionally allowed regions of the Ramachandran plot according to
PROCHECK (21). The figures were prepared with MOLSCRIPT (22) and
Raster3D (23).
Overview of the Structure and Physiological Relevance--
The
N-terminal region of p67phox used in this
study was shown to be fully competent in NADPH oxidase activation both
in vitro and in vivo (24, 25). The electron
density map obtained from SAD phasing was clearly interpretable (Fig.
1).
p67phox-(1-213) consists of four TPR motifs
followed by an extended loop, which inserts into the hydrophobic groove
formed by the helical organization of the TPRs (Fig.
2). The first three TPRs are contiguous and 16 residues (105 to 120) forming two antiparallel
The recent structure of Rac-p67phox-(1-203)
(12) showed that all the residues of p67phox
involved in the complex formation belong to the inserted Mutations in p67phox That Cause Chronic
Granulomatous Disease--
CGD is an inherited disorder of neutrophil
function characterized by an increased susceptibility to infection
because of a defect in the NADPH oxidase components. Mutations
involving p67phox are single cases and are
located in the N-terminal region of the protein. An in-frame deletion
of K58 was found to prevent Rac binding to
p67phox (28). Deletion of amino acids 19-21
renders the protein inefficient for oxidase activation (29). These
amino acids are located between helices A and B of TPR1 and are exposed
to solvent. Various point mutations encountered in
p67phox of CGD patients (R77Q, G78E, A128V,
D160V/K161E) were studied and shown to be associated with the absence
of the protein in vivo (6, 30). This observation accounts
either for mRNA or protein instability or for an increased
sensitivity to proteases. Amino acids Gly-78 and Ala-128 are located in
similar positions in the
We produced G78E and A128V CGD mutants of
p67phox-(1-213). The G78E mutant was
insoluble in bacteria. Because the A128V mutant showed lower solubility
than the native protein, it was expressed at 15 °C instead of
37 °C. Surprisingly, this mutant was still able to bind Rac and to
activate the NADPH oxidase in a cell-free system at 25 °C (Fig.
3), suggesting correct folding. Moreover, the CD spectra at 25 °C of the mutant and of the native proteins were identical and characteristic of
The behavior of the two CGD mutants can be interpreted with respect to
the structure of the native protein
p67phox-(1-213). The mutation G78E
leads to a misfolding of the protein by steric hindrance within the
TPR2 motif. In the less drastic mutant A128V, Val-128 would be located
at 2.2 Å of the carbonyl of Ala-140 or of the hydroxyl of Tyr-172
(Fig. 6). This hydroxyl in the native
structure is stabilized by two hydrogen bonds. Although the environment
should be able to accommodate this moderate steric change, the
hydrogen-bond network is perturbed and the structure of the A128V
mutant is weakened. Within the cellular environment at 37 °C, the
decrease in stability because of this mutation probably leads to
misfolded p67phox protein. This mutation is
akin to other point mutations introduced at position 8 of the A helix
of a TPR motif in p62cdc23 (Ala
Thus, the structure of the active N-terminal domain of
p67phox-(1-213) highlights a short
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
helices termed A and B and a TPR domain (a succession of TPR motifs) adopts an overall
superhelical fold (9). Using various binding assays, a first study
suggested that p67phox interacts with Rac
only through 30 residues downstream of the TPR domain (10). A second
study showed that Rac-p67phox interaction
involves the TPR domain itself (11). To unravel the properties and Rac
binding sites on p67phox, structures of the
non-complexed p67phox and of the
Rac-p67phox complex had to be deciphered.
Recently, a first piece of the puzzle was achieved with the
structure at 2.4 Å of the complex between the N-terminal region of
p67phox (residues 1-203) and Rac-GTP,
showing an unusual interaction of a TPR domain with a partner protein
(12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-S in the presence of 4 mM EDTA,
followed by addition of MgCl2 to 20 mM. An
optimal amount of arachidonic acid (5-20 nmol) was added with strong
agitation. After a 10-min activation, the elicited oxidase activity was
assessed using the superoxide dismutase inhibitable cytochrome
c reduction in the presence of 250 µM NADPH
and 100 µM cytochrome c, followed at 550 nm
using a Labsystem IEMS microplate reader.
-S on the beads. The beads were
washed and incubated for 2 h at 4 °C with a stoichiometric
amount of p67phox-(1-213) or the A128V
mutant. After washing, proteins bound to the beads were analyzed by
SDS-polyacrylamide gel electrophoresis.
Data reduction and phasing statistics
Refinement statistics
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-strands are
inserted between TPR3 and TPR4. A comparison with other recently reported TPR domain structures (9, 26) highlights a remarkable conservation of the overall structure. For example, the TPR1 domain of
Hop (PDB accession code 1ELW), composed of three TPR motifs, could be
superimposed on three consecutive TPR motifs of
p67phox with rmsd values of 0.95 Å and 1.40 Å with the first three and the last three
p67phox TPRs, respectively. This indicates
that the fold of TPR domains is highly conserved despite a rather small
sequence identity; in addition, the insertion of amino acids 105-120
in p67phox does not disrupt the superhelical
structure of the TPR domain. Following TPR4, a helix (residues
156-166) terminates the TPR domain and an extended structure (residues
168-186) folds back into the internal hydrophobic groove of the
super-helix. The structure ends with a short helix (residues 187-193),
mainly composed of polar residues. Residues 170-185 interact
extensively with residues belonging to the A helices of TPR motifs or
to the inserted
-strands (Fig. 2b), probably stabilizing
the overall structure. In particular, Arg-181 interacts with Gln-115.
In the Rac-p67phox complex, Arg-181 is
replaced by Lys-181 and the same type of interaction is observed,
consistent with the existence of a polymorphism (6). The sequence of
p67phox (1) is extremely rich in basic
amino acids with 22 lysine and 6 arginine residues located mainly on
the external surface of the super-helix. These residues are uniformly
distributed at the surface without forming a highly basic patch.
Residues downstream from Leu-193 are not visible in the electron
density map.
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Fig. 1.
Experimental electron density.
The experimental map at 1.8 Å resolution (contoured at 1 )
is clearly interpretable and side chains are easily identified.
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Fig. 2.
Overall structure. A ribbon
representation of the structure shows the four TPR motifs labeled in
a, the A and B type helices are shown in b. The
TPRs adopt an overall super-helix fold (colored from blue to
green). The C terminus in red is in an extended
conformation locked in the hydrophobic groove formed by the TPRs and
ends with a short helix. The inserted -strands between TPR 3 and 4 are shown in yellow. a and b are two
perpendicular views of the molecule.
-strands (Arg-102, Asn-104, Leu-106, Asp-108) or to the loops connecting TPR1 to
TPR2 (S37) and TPR2 to TPR3 (D67, H69). The structures of both the
non-complexed and complexed forms of p67phox
can be remarkably well superimposed with a rmsd of 0.57 Å on main
chain atoms, showing that the interaction of
p67phox (1) with Rac does not require
structural rearrangements of this N-terminal region. In particular, the
residues involved in the interaction with Rac are totally accessible in
the non-complexed structure, their side chains pointing toward the
solvent. Although the two constructs of
p67phox (1-203 in the complex and 1-213 in
this study) differ only by ten amino acids in length, the first was
reported to be inactive whereas the second fully activates NADPH
oxidase in vitro (24). This drastic difference could be
related to the presence of the C-terminal
-helix (residues 187-193)
present in our model that is not seen in the
Rac-p67phox complex structure, although
amino acids 1-203 of p67phox are present in
the crystal. The folding of this helix is probably facilitated by the
presence of amino acids up to 213. From functional studies of various
truncated forms of p67phox, amino acids
199-210 of p67phox were defined as an NADPH
oxidase activation domain (24), and this region was reported to be
involved in the regulation of electron transfer (27). The comparison of
the structure of p67phox-(1-213) to that of
p67phox-(1-203) in the
Rac-p67phox complex (12) suggests that the
activation domain includes helix 187-193.
-helices of the TPR motifs (position 8 of
helix A). Their mutations are likely to destabilize TPR packing. A
sequence alignment of multiple TPRs shows that position 8 in
helix A is restricted to glycine, alanine, or serine residues.
-helices, indicating no modification in the secondary structure (Fig.
4, inset). Altogether, these
experiments do not explain the CGD phenotype of this mutant. To assess
slight differences in the tertiary structure, the native protein and
the A128V mutant were subjected to limited proteolysis by trypsin at
25 °C. As shown in Fig. 5, the native
protein is poorly degraded up to 60 min, whereas the A128V mutant shows
notable degradation starting at 15 min. Additionally, the CD spectrum as a function of the temperature shows that the native protein is still
stable at 40 °C whereas the A128V mutant begins to loose its helical
folding at 30 °C (Fig. 4) and precipitates at 40 °C.
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Fig. 3.
Functional assays of the A128V mutant.
a represents the amount of superoxide anion per min and per
mg of membrane protein as a function of
p67phox. The activity of the native
p67phox-(1-213) and of the A128V mutant are
shown with squares and full circles,
respectively. The amounts of p47phox and Rac
were constant during each test. The SDS-polyacrylamide gel
electrophoresis in b shows the binding of the native and the
mutant proteins to Rac as described under "Experimental
Procedures." No binding was observed when GST alone was
present.
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Fig. 4.
CD Spectra. CD spectra at 15 °C
(solid line) and 35 °C (dashed line) of the
A128V mutant. The inset shows the molar ellipticity at 222 nm for the native protein (circles, solid line) and for the
A128V mutant (squares, dashed line) as a function of the
temperature.
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Fig. 5.
SDS-polyacrylamide gel after tryptic
digestion of p67phox-(1-213). The
lanes represent the native protein and the A128V mutant at
0, 15, 30, and 60 min digestion.
Thr or Gly
Asp), which
also confer thermolability to the protein (31).
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Fig. 6.
Environment of Ala-128. The figure shows
the interactions of the extended C terminus (red) with TPR4
(green). Ala-128 is tightly packed in this environment.
Valine at position 128 (light gray) is superimposed on
alanine.
-helix that participates to the NADPH oxidase activation domain.
Interestingly, most of the CGD mutants of
p67phox are located in the N-terminal region
of p67phox. The structural and functional
studies reported here permit an elucidation of the instability of the
G78E and A128V mutants and may be extended to mutations that affect TPR
folding. The coordinates and the structure factors have been deposited
in the Protein Data Bank with ID code 1hh8.
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ACKNOWLEDGEMENTS |
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We thank A. Thompson, M. Roth, G. Leonard, D. Fleury, and S. Malbet for help with data collection at ESRF, M. Picard for technical assistance, J. P. Pichon for mass spectroscopy analysis, D. Madern for CD measurements, and P. V. Vignais for stimulating discussions and critical reading of the manuscript.
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FOOTNOTES |
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* This work was supported in part by the Association pour la Recherche sur le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
The atomic coordinates and the structure factors (code 1hh8) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).
¶ To whom correspondence should be addressed. Tel.: 33-0-4-3878- 9583; Fax: 33-0-4-3878-5494; E-mail: pebay@ibs.fr.
Published, JBC Papers in Press, March 21, 2001, DOI 10.1074/jbc.M100893200
2 A. Royant, S. Grizot, F. Fieschi, E. M. Landau, R. Kahn, and E. Pebay-Peyroula, manuscript in preparation.
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ABBREVIATIONS |
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The abbreviations used are:
CGD, chronic
granulomatous disease;
TPR, tetratricopeptide repeat;
SAD, single
wavelength anomalous dispersion;
rmsd, root mean square deviation;
GST, glutathione S-transferase;
GTP--S, guanosine
5'-3-O-(thio)triphosphate.
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