Adrenodoxin Reductase-Adrenodoxin Complex Structure Suggests Electron Transfer Path in Steroid Biosynthesis*

Jürgen J. MüllerDagger §, Anna LapkoDagger , Gleb Bourenkov||, Klaus RuckpaulDagger , and Udo HeinemannDagger **DaggerDagger

From Dagger  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



    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
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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.



    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES

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 (P45011beta , 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.

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.


    EXPERIMENTAL PROCEDURES
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INTRODUCTION
EXPERIMENTAL PROCEDURES
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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 epsilon -amino group of Lys66 in Adx and the gamma -carboxyl group of Glu4 in AR.

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).


                              
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Table I
Crystallographic data and structure refinement

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.


    RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
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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 Calpha atoms with a root mean square deviation (r.m.s.d.) of 0.46 Å and of the AR Calpha 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).

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.



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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).

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.



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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).

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.



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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 1sigma . 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 1sigma . The cross-linked side chains are 90° clockwise rotated around the long axis.

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-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.

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.



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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.

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.


    ACKNOWLEDGEMENTS

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.


    FOOTNOTES

* 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.

Dagger Dagger 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.


    ABBREVIATIONS

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES


1. Bernhardt, R. (1996) Rev. Physiol. Biochem. Pharmacol. 127, 137-221[Medline] [Order article via Infotrieve]
2. Vickery, L. E. (1997) Steroids 62, 124-127[CrossRef][Medline] [Order article via Infotrieve]
3. Grinberg, A. V., Hannemann, F., Schiffler, B., Müller, J. J., Heinemann, U., and Bernhardt, R. (2000) Proteins Struct. Funct. Genet. 40, 590-612[CrossRef][Medline] [Order article via Infotrieve]
4. Lambeth, J. D., Seybert, D. W., Lancaster, J. R., Salerno, J. C., and Kamin, H. (1982) Mol. Cell. Biochem. 45, 13-31[Medline] [Order article via Infotrieve]
5. Kido, T., and Kimura, T. (1979) J. Biol. Chem. 254, 11806-11815[Abstract]
6. Hara, T., and Takeshima, M. (1994) in Cytochrome P450, 8th International Conference (Lechner, M. C., ed) , pp. 417-420, Libbey Eurotext, Paris
7. Müller, A., Müller, J. J., Muller, Y. A., Uhlmann, H., Bernhardt, R., and Heinemann, U. (1998) Structure 6, 269-280[Medline] [Order article via Infotrieve]
8. Pikuleva, I., Tesh, K., Waterman, M. R., and Kim, Y. (2000) Arch. Biochem. Biophys. 373, 44-55[CrossRef][Medline] [Order article via Infotrieve]
9. Ziegler, G. A., Vonrhein, C., Hanukoglu, I., and Schulz, G. E. (1999) J. Mol. Biol. 289, 981-990[CrossRef][Medline] [Order article via Infotrieve]
10. Lapko, A., Müller, A., Heese, O., Ruckpaul, K., and Heinemann, U. (1997) Proteins Struct. Funct. Genet. 28, 289-292[CrossRef][Medline] [Order article via Infotrieve]
11. Hara, T., and Kimura, T. (1989) J. Biochem. (Tokyo) 105, 594-600[Abstract]
12. Hara, T., and Miyata, T. (1991) J. Biochem. (Tokyo) 110, 261-266[Abstract]
13. Otwinowski, Z., and Minor, W. (1997) Methods Enzymol. 276, 307-326
14. Navaza, J. (1994) Acta Crystallogr. Sect. A 50, 157-163[CrossRef]
15. Brünger, A. T., Adams, P. D., Clore, G. M., DeLano, W. L., Gros, P., Grosse-Kunstleve, R. W., Jiang, J.-S., Kuszewski, J., Nilges, M., Pannu, N. S., Read, R. J., Rice, L. M., Simonson, T., and Warren, G. L. (1998) Acta Crystallogr. Sect. D Biol. Crystallogr. 54, 905-921[CrossRef][Medline] [Order article via Infotrieve]
16. Laskowski, R. A., MacArthur, M. W., Moss, D. S., and Thornton, J. M. (1993) J. Appl. Crystallogr. 26, 283-291[CrossRef]
17. Kraulis, P. J. (1991) J. Appl. Crystallogr. 24, 946-950[CrossRef]
18. Hanukoglu, I. J. (1992) Steroid Biochem. Mol. Biol. 43, 779-804
19. Ishimura, K., and Fujita, H. (1997) Microsc. Res. Tech. 36, 445-453[CrossRef][Medline] [Order article via Infotrieve]
20. Evans, S. V. (1993) J. Mol. Graphics 11, 134-138[CrossRef][Medline] [Order article via Infotrieve]
21. Lambeth, J. D., Geren, L. M., and Millet, F. (1984) J. Biol. Chem. 259, 10025-10029[Abstract/Free Full Text]
22. Coghlan, V. M., and Vickery, L. E. (1992) J. Biol. Chem. 267, 8932-8935[Abstract/Free Full Text]
23. Coghlan, V. M., and Vickery, L. E. (1991) J. Biol. Chem. 266, 18606-18612[Abstract/Free Full Text]
24. Brandt, M. E., and Vickery, L. E. (1993) J. Biol. Chem. 268, 17126-17130[Abstract/Free Full Text]
25. Honig, B., and Nicholls, A. (1995) Science 268, 1144-1149[Medline] [Order article via Infotrieve]
26. Nicholls, A., Sharp, K. A., and Honig, B. (1991) Proteins Struct. Funct. Genet. 11, 281-296[Medline] [Order article via Infotrieve]
27. Pochapsky, T. C., Lyons, T. A., Kazanis, S., Arakaki, T., and Ratnaswamy, G. (1996) Biochimie (Paris) 78, 723-733[CrossRef][Medline] [Order article via Infotrieve]
28. Müller, J. J., Müller, A., Rottmann, M., Bernhardt, R., and Heinemann, U. (1999) J. Mol. Biol. 294, 501-513[CrossRef][Medline] [Order article via Infotrieve]
29. Hurley, J. K., Weber-Main, A. M., Stankovich, M. T., Benning, M. M., Thoden, J. B., Vanhooke, J. L., Holden, H. M., Chae, Y. K., Xia, B., Cheng, H., Markley, J. L., Martinez-Julvez, M., Gomez-Moreno, C., Schmeits, J. L., and Tollin, G. (1997) Biochemistry 36, 11100-11117[CrossRef][Medline] [Order article via Infotrieve]
30. Page, C. C., Moser, C. C., Chen, X., and Dutton, P. L. (1999) Nature 402, 47-52[CrossRef][Medline] [Order article via Infotrieve]
31. Beratan, D. N., Betts, J. N., and Onuchic, J. N. (1991) Science 252, 1285-1288[Medline] [Order article via Infotrieve]


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