From Max-Delbrück-Centrum für Molekulare
Medizin, Robert-Rössle-Strasse 10, D-13125 Berlin,
MPG-ASMB, c/o DESY, Notkestrasse 85, D-22603 Hamburg, and
** Institut für Chemie-Kristallographie, Freie
Universität Berlin, Takustrasse 6, D-14195 Berlin, Germany
Received for publication, September 18, 2000, and in revised form, October 25, 2000
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ABSTRACT |
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The steroid hydroxylating system of adrenal
cortex mitochondria consists of the membrane-attached
NADPH-dependent adrenodoxin reductase (AR), the soluble
one-electron transport protein adrenodoxin (Adx), and a
membrane-integrated cytochrome P450 of the CYP11 family. In the 2.3-Å
resolution crystal structure of the Adx·AR complex, 580 Å2 of partly polar surface are buried. Main
interaction sites are centered around Asp79,
Asp76, Asp72, and Asp39 of Adx and
around Arg211, Arg240, Arg244, and
Lys27 of AR, respectively. In particular, the region around
Asp39 defines a new protein interaction site for Adx,
similar to those found in plant and bacterial ferredoxins. Additional
contacts involve the electron transfer region between the redox centers of AR and Adx and C-terminal residues of Adx. The Adx residues Asp113 to Arg115 adopt 310-helical
conformation and engage in loose intermolecular contacts within a deep
cleft of AR. Complex formation is accompanied by a slight domain
rearrangement in AR. The [2Fe-2S] cluster of Adx and the
isoalloxazine rings of FAD of AR are 10 Å apart suggesting a possible
electron transfer route between these redox centers. The AR·Adx
complex represents the first structure of a biologically relevant
complex between a ferredoxin and its reductase.
In mitochondria of the adrenal cortex, the cytochrome P450 enzymes
of the CYP11 family catalyze the side chain cleavage of cholesterol to
form pregnenolone (P450scc,1
CYP11A1) and are involved in the formation of cortisol (P45011 Recently, the crystal structures of two forms of bovine adrenodoxin (7,
8) and of adrenodoxin reductase (9) were determined. These structures
revealed the general topology of the two proteins and the molecular
environments of the [2Fe-2S] cluster of Adx and the FAD moiety of AR.
Here, we report the 2.3-Å resolution crystal structure of a
cross-linked 1:1 complex of full-length Adx and AR. This structure
shows the geometry of an electron transfer complex of soluble, freely
dissociable proteins from a higher eukaryote for the first time,
highlights structural adaptations that accompany the binding of AR to
Adx, and permits us to predict electron transport paths in their complex.
Sample Preparation--
Recombinant bovine Adx and AR were
purified and crystallized as described (10). The synthesized Adx
differs from the wild-type protein by the exchange of Ser1
for glycine and is composed of 128 amino acids, including the N- and
C-terminal residues missing in the truncated adrenodoxin, Adx-(4-108), studied earlier (7). Cross-linking of AR to Adx has also been described (11, 12). The native complex is formed at low
ionic strength between the two proteins, and the cross-linking was
carried out with the water-soluble coupling reagent
1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC, purchased from
Sigma). The mixture of Adx (1.2 µmol) and AR (300 nmol) was dialyzed
for 18-20 h against 20 mM potassium phosphate, pH 7.2, followed by addition of an equal volume of fresh 8 mM EDC
solution in distilled water and incubation at 4 °C in the dark with
occasional stirring. After 8 h, excess of the reagent was removed
on a Sephadex G-25 column equilibrated with 10 mM potassium
phosphate, pH 7.4. The colored fraction was pooled and applied on a
2.4 × 10 cm DEAE-Fractogel column and washed with two gradient
solutions as follows: 10-50 mM potassium phosphate, pH
7.4, for 3 h; 50-100 mM potassium phosphate, pH 7.4, for 3 h. The peak containing the covalent cross-linked complex of
the recombinant Adx and AR was consequently purified on an AD-Sepharose
column to remove residual AR and on an ADP-Sepharose column to remove
unbound Adx. The cross-linking of AR to Adx with EDC was expected (11,
12) to yield an amide bond between the X-ray Data Collection--
Four x-ray diffraction data sets from
three crystals were collected at 100 K on MAR345 imaging plates at beam
lines BW7B (EMBL Outstation at DESY, Hamburg) and BW6 (MPG-ASMB, c/o
DESY). Due to problems with spatial reflection overlaps caused by the
long c axis of 607.85 Å, the data reached only 79%
completeness (85% at 2.5 Å resolution), although several detector
settings were used. The data sets were processed by DENZO/SCALEPACK
(13) and contained 55,229 unique reflexes after merging (Table
I).
Structure Determination and Refinement--
The structure of the
AR·Adx complex was solved by molecular replacement using the
coordinates of AR and Adx-(4-108) as deposited in the Protein Data
Bank (codes 1cjc and 1ayf). Two complexes per asymmetric unit with a
molecular mass of 64,927 Da each were assembled by placing the
protein molecules into the unit cell using AMORE (14) and rigid-body
refinement with the program CNS (15) resulting in
R = 0.386, Rfree = 0.394 at 2.8 Å resolution. Application of a solvent mask, positional and atomic
temperature factor refinement, several rounds of manual density
fitting, and the addition of 5 sulfate ions and 277 water molecules
reduced R to 0.223 and Rfree to 0.268 (Table I). 1132 residues out of 1176 could be localized within the
electron density. Almost all modeled water positions are also occupied
in the crystal structures of Adx-(4-108) (7) and AR (9). The averaged
main chain and side chain parameters are equal or better than those in
a set of 118 structures used by PROCHECK (16), and the Ramachandran diagram is free of outliers.
Architecture of the AR·Adx Complex--
The hexagonal crystals
used in this analysis are formed by cross-linked 1:1 complexes between
AR and Adx, both in their oxidized form. Two complexes related by a
noncrystallographic screw rotation are present in the asymmetric unit.
Complex I contains residues 5-117 of Adx and 4-460 of AR, and complex
II contains residues 5-110 of Adx and 5-460 of AR. Electron density
for both complexes clearly reveals the [2Fe-2S] cluster of Adx and
the FAD moiety of AR. As in the crystal structure of the free protein
(9), no NADP is bound to AR. With the exception of a small domain
rearrangement in AR (see below), protein conformation in these
independent copies of the complex is generally similar allowing least
squares superpositions of the Adx C
By fitting the globular Adx molecule into a prominent depression on the
AR surface, a compact AR·Adx complex is formed (Fig. 1). A total of 580 Å2 of
solvent-accessible surface are buried between the protein molecules.
This AR-Adx interface contains many polar residues. In the complex, Adx
contacts both AR domains. In a primary interaction region, polar
contacts are formed between residues of the NADP domain of AR and the
Adx side chains belonging to the interaction domain (7) of the protein.
Further polar interactions take place in a secondary interaction region
where the core domain (7) of Adx contacts the FAD domain of AR and the
covalent cross-link is formed linking Adx Asp39 with AR
Lys27. In a third interaction region, the C-terminal
polypeptide stretch of Adx dips into a deep cleft between the two
globular domains of AR. Adx residues Asp113 to
Arg115 of this region adopt 310-helical
conformation. This contact is assumed to be rather loose, since the
C-terminal residues of Adx adopt high atomic displacement factors up to
80 Å2 indicating flexibility, and the interaction is not
observed in complex II of the crystal where no electron density is seen
beyond Ala110 of Adx. Finally, further hydrogen bonding and
van der Waals interactions are observed between residues bridging the
[2Fe-2S] cluster of Adx and the isoalloxazine ring of the FAD of
AR.
Two electron density peaks near the AR N terminus were assigned as
sulfate ions. Since AR is a membrane-associated protein (18, 19), it is
tempting to speculate that these sulfate positions mark interaction
sites of AR with phospholipids in the membrane. This hypothesis is
supported by the observed arrangement of hydrophobic (e.g.
Trp420) and basic residues (Arg31,
Arg70, Arg73, Lys411,
Lys429, and Arg456) around the sulfate ions
that might interact with lipid or phosphate moieties of the membrane.
Reorientation of AR Domains during Complex Formation--
Both Adx
and AR are two-domain proteins. Adx consists of a core domain
containing the [2Fe-2S] cluster and a small interaction domain (7),
and AR contains a FAD domain and a NADP domain of about equal size (9).
Whereas no significant difference between Adx-(4-108) and Adx as
present in the complex is detected, the two AR domains show a slightly
different orientation with respect to each other when the complex is
compared with free AR (Fig. 2). After
superposition of the FAD domains of AR (r.m.s.d. = 0.38 Å), optimal
fit of the NADP domains requires a 3.7° rotation (7.2° for complex
II). Considering Arg240 and Lys27 of AR as
reference contact points with Adx (see below), this domain
reorientation results in a narrowing of the distance between these two
anchor points by 2.4 Å (4.2 Å in complex II). AR thus has the ability
to adapt to the Adx molecule in the binary complex by domain
reorientation to various degrees.
Complex Formation by Electrostatic Interactions--
The AR·Adx
complex displays a highly charged surface (Fig.
3, top) arising from
interacting surfaces that are predominantly acidic (Adx) or basic (AR).
Of the 580 Å2 of solvent-accessible surface buried in the
complex, 325 Å2 are from hydrophobic side chains. Nearly
half of the AR-Adx interface is composed of polar and charged residues
engaging in a large number of hydrogen bonds and salt links. Hence,
electrostatic interactions may be considered the primary driving force
for complex formation in agreement with chemical modification (21) and
site-directed mutagenesis experiments (1, 22-24) of AR and Adx.
Electrostatic interactions predominate in the two main interaction
sites of the AR·Adx complex. In the primary interaction region (Fig.
3, bottom left), arginines 211, 240, and 244 of the NADP domain of AR are involved in numerous salt bridges with Adx carboxylate groups. Aspartates 72, 76, and 79 of the Adx interaction domain are binding partners to AR, whereas the acidic residues Glu73 and Glu74 of Adx are facing away from the
interface. The electron density provides no evidence for a covalent
cross-link formed at this interaction site. Acidic Adx residues located
at the primary AR-Adx interaction region are known also to be involved
in cytochrome P450scc binding (1). Given the participation of several
of these side chains in contacts to AR, the formation of an organized 1:1:1 complex between AR, Adx, and cytochrome P450scc for electron transport during steroid biosynthesis must be regarded as very unlikely.
A secondary interaction region is centered around the Adx residues
Asp39 and Asp41 contacting His28
and Lys27 of AR, respectively (Fig. 3, bottom
right). Again, these contacts are polar and are mainly
formed by charged side chains. Asp39 and Asp41
are located in the core domain of Adx at a surface region that has been
implicated in cytochrome P450cam (CYP101) binding by the homologous
putida redoxin (27). An involvement of Asp39 in
redox-partner binding has been suggested earlier (28) based on a
comparison of the Adx structure with crystal structures of plant-type
ferredoxins (29).
Covalent cross-linking of Adx and AR with carbodiimide prior to
crystallization results in the formation of a peptide bond between the
carboxylate function of Asp39 and the primary amino group
of Lys27 as clearly revealed by electron density (see Fig.
3, bottom right). This finding is unexpected, since the
cross-linking procedure employed was reported (11, 12) to yield a
covalent bond linking AR Glu4 and Adx Lys66.
Peptide sequencing and mass spectrometric analysis prove that the
Glu4-Lys66 cross-link is indeed not formed in
the AR·Adx complex.2
The suggested Glu4-Lys66 cross-link is
incompatible with the binding mode to AR of Adx reported here. It does
not permit contacts between the proteins in the primary interaction
region as supported by mutagenesis experiments (1, 22-24) and renders
unlikely a close enough approach of the redox centers for electron
transfer. For these reasons we are convinced that the reported complex, and not a complex cross-linked at AR Glu4/Adx
Lys66, represents the functional interaction between AR and
Adx.
Possible Electron Transfer Path--
Efficient electron transfer
between the redox centers requires spatial proximity. The closest
approach of atoms belonging to the [2Fe-2S] cluster of Adx and the
isoalloxazine ring of the FAD of AR is 10.3 Å (9.65 Å in complex II),
well within the 14-Å threshold reported to define the limit of
electron tunneling in a protein medium (30). The fractional packing
density of protein groups between the redox centers is 0.61 (0.73 in
complex II), again within the observed range of densities found in
natural multiredox center oxidoreductases of known structure (30).
Thus, from proximity and packing density considerations alone, one may conclude that the geometry of the AR·Adx complex will support electron tunneling between the redox centers. The observed geometry is
calculated by ETUNNEL (30) to support electron transfer rates of
108 to 109 s
The program HARLEM, analyzing distinct protein structures with respect
to tunneling probabilities (31), was further used to compute possible
electron transfer routes between AR and Adx (Fig.
4). According to this analysis, electrons
would most likely travel along covalent bonds, requiring two
through-space jumps from the FAD isoalloxazine to AR Ile376
and from AR Thr377 to Adx Cys52, one of the
[2Fe-2S] ligands. However, alternative transfer paths and a possible
involvement of water molecules located at the interface region cannot
be ruled out.
In summary, the 2.3-Å crystal structure of the AR·Adx redox complex
suggests modes of electron transfer between a soluble [2Fe-2S]
ferredoxin and its cognate reductase. It reveals the importance of
electrostatic interactions in complex formation, in agreement with the
concept of "electrostatic steering" (1, 7), and demonstrates that a
slight domain rearrangement in AR is required for a tight AR-Adx interaction.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
, CYP11B1) and aldosterone (P450aldo, CYP11B2) (1). The enzymatic activity of the cytochrome P450-dependent steroid
hydroxylases is based on their ability to activate molecular oxygen by
reductive splitting of dioxygen. This multistep reaction requires the
transfer of electrons from the flavoprotein adrenodoxin reductase (AR) via adrenodoxin (Adx) to the terminal cytochromes P450 as electron acceptors in dependence on the specific hydroxylation substrate (1-3).
Several models for electron transfer have been discussed, including a
shuttle model in which Adx forms consecutive 1:1 complexes (4) with AR
and cytochrome P450scc and models requiring the formation of an
organized 1:1:1 ternary complex (5) or a 1:2:1 quaternary complex (6)
between AR, Adx, and cytochrome P450scc. Common to these models is a
complex between AR and Adx during the first steps of electron transfer
from the reductase to the cytochrome P450.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
-amino group of
Lys66 in Adx and the
-carboxyl group of Glu4
in AR.
Crystallographic data and structure refinement
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
atoms with a root mean square
deviation (r.m.s.d.) of 0.46 Å and of the AR C
positions with a
r.m.s.d. of 0.95 Å. The description of the crystal structure of the
AR·Adx complex will focus on complex I, where the C terminus of Adx
is ordered up to Ser117, revealing a number of residues
neither observed in the structure of Adx-(4-108) (7) nor in that of
full-length Adx (8).
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Fig. 1.
Crystal structure of the AR·Adx
complex. AR-Adx contacts occur at the primary and secondary
interaction regions and the region between the [2Fe-2S] cluster of
Adx and the isoalloxazine ring of the FAD of AR. C-terminal residues of
Adx are also in contact with AR. The side chains of some residues
involved in polar AR-Adx interactions are displayed. For close-ups of
the contact sites see Figs. 3 and 4. The brown triangle
marks the position of Adx Lys66 and the green
marks AR Glu4, both residues maintaining another cross-link
reported recently (11, 12). Figure was produced with MOLSCRIPT
(17).
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Fig. 2.
Least squares superposition of the FAD
domains of AR (bottom) from the crystal structures of
free AR (9) (black) and the AR·Adx complex (molecule
1 in red and molecule 2 in
gold). In the complex, the NAD domains of AR
(top) undergo a slight domain rearrangement relative to the
FAD domains that pulls the regions in contact with Adx closer together.
Arg211, Arg240, and Arg244 of AR
are part of the primary and Lys27 is part of the secondary
interaction region. This figure and Figs. 3 (bottom) were
drawn with SETOR (20).
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Fig. 3.
Electrostatic interactions between AR and
Adx. Top, surface drawings of AR (right),
the AR·Adx complex in the orientation displayed in Fig. 1
(center), and Adx (left). Adx and AR are rotated
relative to their orientation in the complex as indicated to emphasize
the interacting surfaces. Surfaces are colored corresponding to the
electrostatic potential calculated by the program DELPHI (25) for an
ionic strength of 0.1 M. The deepest shades of
blue and red correspond to potentials of ± 10 kT. Blue surface regions carry positive
charge, and red surfaces are negatively charged. In the
primary and secondary interaction sites, predominantly positively
charged surface areas of AR are brought into close contact with
predominantly negatively charged regions of the Adx surface. Surfaces
were calculated and displayed with GRASP (26). Bottom left,
salt bridges (dotted lines) connecting AR and Adx in the
primary interaction region. Residues are labeled black in AR
and red in Adx. Bottom right, secondary
interaction region with brown colored 2Fo Fc electron density contoured at 1
. A salt
bridge linking His28 of AR and Asp41 of Adx is
indicated by the dotted line, and water molecules are shown
as blue spheres. Note the covalent cross-link between AR
Lys27 and Adx Asp39. The inset shows
the green colored 2Fo
Fc omit map, contoured at 1
. The cross-linked
side chains are 90° clockwise rotated around the long axis.
1. This
is orders of magnitude above the experimentally determined (4)
flavin-to-iron transfer rate of 3-4 s
1. By
assuming that the covalent cross-link does not force an unnaturally tight AR-Adx interaction, it may thus be concluded that the rate of the
redox reaction in which AR and Adx are involved is not limited by
electron transfer within the AR·Adx complex.
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Fig. 4.
Electron transfer region between the
[2Fe-2S] cluster of Adx and the FAD moiety of AR. The
hypothetical electron pathway shown in red was calculated
with the program HARLEM (31). Red dotted lines mark
through-space electron jumps. The AR-Adx interface is stabilized by
hydrogen bonds (blue dotted lines) and van der Waals
contacts. Residues are labeled black for AR and
red for Adx. The blue spheres are water
molecules.
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ACKNOWLEDGEMENTS |
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We are grateful to C. Jung and Y. A. Muller (Max-Delbrück-Centrum für Molekulare Medizin) for critically reading the manuscript and R. Bernhardt (Universität des Saarlandes) for numerous helpful discussions. We thank also D. N. Beratan for the electron pathway calculation program HARLEM.
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FOOTNOTES |
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* This work was supported by Deutsche Forschungsgemeinschaft Grants He 1318/19-1 and WER436 and the Fonds der Chemischen Industrie.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 1e6e) 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 may be addressed. Tel.: 49 30 9406 3421; E-mail: JJM@MDC-Berlin.de.
¶ On leave from the International Sakharow Institute of Radioecology, 2220009 Minsk, Belarus.
To whom correspondence may be addressed. Tel.: 49 30 9406-3420;
E-mail: Heinemann@MDC-Berlin.de.
Published, JBC Papers in Press, October 25, 2000, DOI 10.1074/jbc.M008501200
2 E.-Ch. Müller, A. Lapko, A. Otto, J. J. Müller, K. Ruckpaul, and U. Heinemann, unpublished results.
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ABBREVIATIONS |
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The abbreviations used are: P450scc, cytochrome P450scc (CYP11A1); Adx, bovine adrenodoxin; Adx(4-108), truncated Adx; AR, adrenodoxin reductase; EDC, 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide; r.m.s.d., root mean square deviation.
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