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Originally published In Press as doi:10.1074/jbc.M410073200 on October 8, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54750-54758, December 24, 2004
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Crystal Structure of the Kelch Domain of Human Keap1*

Xuchu Li{ddagger}, Donna Zhang§, Mark Hannink{ddagger}§, and Lesa J. Beamer{ddagger}

From the {ddagger}Department of Biochemistry and the §Life Sciences Center, University of Missouri-Columbia, Columbia, Missouri 65211

Received for publication, September 1, 2004 , and in revised form, October 4, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Keap1 is a substrate adaptor protein for an ubiquitin ligase complex that targets the Nrf2 transcription factor for degradation. Keap1 binds Nrf2 through its C-terminal Kelch domain, which contains six copies of the evolutionarily conserved kelch repeat sequence motif. The structure of the Kelch domain from human Keap1 has been determined by x-ray crystallography to a resolution of 1.85 Å. The Kelch domain forms a 6-bladed {beta}-propeller structure, with residues at the C terminus forming the first strand in the first blade. Key structural roles have been identified for the highly conserved glycine, tyrosine, and tryptophan residues that define the kelch repeat sequence motif. In addition, we show that substitution of a single amino acid located within a loop that extends out from the bottom of the {beta}-propeller structure abolishes binding of Nrf2. The structure of the Kelch domain of Keap1 represents a high quality model for the superfamily of eukaryotic kelch repeat proteins and provides insight into how disease-causing mutations perturb the structural integrity of the Kelch domain.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Exposure to reactive oxygen species and other chemically reactive molecules can damage biological macromolecules (1). Oxidative damage to biological macromolecules has been implicated in cancer, inflammation, cardiovascular, and neurodegenerative diseases and aging (2–7). Eukaryote cells, which are exposed to reactive oxygen species from both intrinsic and extrinsic sources, have evolved multiple mechanisms to neutralize reactive molecules and minimize oxidative damage. One major mechanism for protection against oxidative damage involves the coordinated induction of a group of cytoprotective genes, including the cancer-preventive Phase 2 stress response genes, which enable cells to neutralize reactive molecules and restore cellular redox homeostasis (8, 9). This cytoprotective genetic program is regulated at the transcriptional level by the transcription factor, Nrf2, which binds to cis-acting DNA sequences termed antioxidant response elements (AREs)1 (8–11).

Nrf2 has a conserved C-terminal basic zipper domain required for both protein-protein interactions and binding to DNA (12). Nrf2 contains a large central transcriptional activation domain that recruits transcriptional co-activators to ARE motifs in the promoter regions of Phase 2 genes (13). Nrf2 also contains a small N-terminal negative regulatory domain, termed Neh2, which mediates binding of Nrf2 to the cytoplasmic Keap1 protein (14). An evolutionarily conserved amino acid sequence motif, ETGE, located within the Neh2 domain of Nrf2, is required for binding of Nrf2 to Keap1 (15).

Compelling genetic and biochemical evidence has implicated the Keap1 protein as the major upstream regulator of Nrf2 (10, 16). Keap1 regulates both subcellular localization and steady-state levels of Nrf2. Keap1 is a cytoplasmic protein that binds actin, and an intact actin-based cytoskeleton is required for Keap1 to sequester Nrf2 in the cytoplasm (17). Keap1 also functions as a substrate adaptor protein to bring Nrf2 into a Cul3-dependent ubiquitin ligase complex (18–20). Keap1 binds to Cul3 via its N-terminal BTB domain (for broad-complex, tramtrack, and bric a brac) and central linker domain, while the C-terminal Kelch domain of Keap1 binds to the Neh2 domain of Nrf2 (14, 19, 20). Under homeostatic conditions of cellular function, this E3 ubiquitin ligase complex, consisting of Nrf2, Keap1, Cul3, Rbx1, and an E2 ubiquitin-conjugating enzyme, targets specific lysine residues within the Neh2 domain of Nrf2 for ubiquitin conjugation and marks Nrf2 for degradation by the 26 S proteosome (18–20). However, upon exposure to oxidative stress, ubiquitination of Nrf2 is inhibited, resulting in increased steady-state levels of Nrf2 and increased transcription of cytoprotective ARE-dependent genes (21).

BTB-Kelch proteins share a common domain organization of an N-terminal BTB domain, a conserved linker domain and a C-terminal Kelch domain. The biological function(s) of the more than 50 BTB-Kelch proteins encoded by the human genome are poorly understood. The few BTB-Kelch proteins that have been characterized have diverse roles in regulation of the cytoskeleton. The Kelch protein of Drosophila melanogaster, the founding member of the BTB-Kelch family, regulates the organization of large actin structures, termed ring canals, which are necessary for intercellular transport of macromolecules in the embryo (22). In contrast, mutations within the human GAN1 protein are the cause of giant axonal neuropathy, a neurological disease characterized by excessive accumulation of intermediate filaments in sensorimotor neurons (23). Several human BTB-Kelch proteins, including GAN1, are able to associate with Cul3 (24). Thus, although Keap1 is the only BTB-Kelch protein that has been demonstrated to function as a substrate adaptor protein, it is likely that the common domain organization of the BTB-Kelch proteins reflects a shared biochemical function that underlies their diverse biological roles.

In this report, we describe the three-dimensional structure of the Kelch domain of human Keap1, as determined by x-ray crystallography. Kelch domains, in general, are comprised of five to seven repeated motifs, ~45–55 amino acids in length, and were first identified in the D. melanogaster Kelch protein (25). Subsequently, the kelch repeat motif was identified in a protein of known structure, the enzyme galactose oxidase from the fungus Dactylium dendroides (26). The kelch repeats in galactose oxidase form a 7-bladed {beta}-propeller, in contrast to members of the mammalian BTB-Kelch family, which typically contain six well-defined kelch repeat motifs. The consensus kelch repeat motif contains several highly conserved residues, including two adjacent glycine residues and a tyrosine/tryptophan pair separated by exactly 7 residues (27, 28). The crystal structure shows that the Kelch domain of Keap1 folds up into a 6-bladed {beta}-propeller structure that uses a C-terminal strand mechanism of closure. The highly conserved tyrosine/tryptophan pairs participate in amino acid contacts involved in interblade interactions, while the glycine doublets participate in a conserved hydrogen-bonding network within each blade. The structure of the Kelch domain of Keap1 represents a high quality model for other members of the BTB-Kelch family and provides significant insight into how disease-causing mutations will perturb the structure of the Kelch domain of GAN1, a human BTB-Kelch protein that is responsible for giant axonal neuropathy (23).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Crystallization and Data Collection—Purification and crystallization of native and selenomethionine (SeMet) Kelch were carried out as previously described (29). Data sets for both the native and SeMet proteins were collected at -160 °C on a Rigaku RU200 rotating anode generator and RAXIS IV detector. Because of the high quality of the SeMet crystals, a highly redundant SeMet data set was collected (Table I) in an attempt to locate the selenium atoms via anomalous scattering using 1.54 Å X-radiation. All data were indexed and integrated with the HKL package (30).


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  		TABLE I
Statistics from the crystallographic analysis

 
Structure Solution and Refinement—The program SOLVE (31) was used with the SeMet data set to locate either seven SeMet sites or the seven SeMet and eight sulfur sites via the anomalous signal, in a Se-SAD approach (32). Although seven SeMet sites (or 14 Se/S sites) were identified, the resulting maps were not readily interpretable, even after density modification. SOLVE was then used with both the native and SeMet data sets, in order to take advantage of both isomorphous and anomalous differences of the SeMet residues (Se-SIRAS). This approach was immediately successful in space group P6522, generating high quality electron density maps (Z-score = 41.6 for seven sites and fom = 0.41 to 1.85 Å resolution), which were further improved through solvent flattening via RESOLVE (fom = 0.70) (33). The automatic chain-tracing feature of RESOLVE built 258 of the 308 residues; the remainder of the model was built interactively using O (34).

Refinement was performed with REFMAC 5.0 (35) using individual, restrained B-factors (Table I). Progress was monitored by use of Rfree, and 5% of the data were set aside for cross-validation before refinement. Water molecules were placed automatically by WATPEAK (36) in peaks greater than 3.0 {sigma} in Fo-Fc maps and within hydrogen bonding distance to nitrogen or oxygen atoms of the protein. The final model of the wild-type protein consists of 288 of the 308 residues, and 313 water molecules. The first 22 residues, 19 of which were from the histidine affinity tag of the protein construct, were not visible in electron density maps. Density for the protein backbone is continuous from residue 322 (the first residue visible at the N terminus) to residue 609 at the C terminus. (Residue numbers refer the amino acid sequence of the intact Keap1 protein, which has 624 residues in total.) Density for the side chains of the following residues was not well defined and they have been truncated to alanine: Gln359, Glu446, His451, Gln528, Glu593, and Arg596. The following side chains have been modeled in two conformations at 50% occupancy: Ser340, Thr388, Cys395, Val411, Val418, Val420, His424, Cys434, Met499, and Val606.

Model Quality—The Kelch model has good geometry, with 91.6% of its residues lying in the most favored regions of the Ramachandran plot (37). The model was also evaluated by SFCHECK (38), and WHAT_ CHECK (39). Figures were prepared with Molscript (40), PYMOL (41), and Raster3D (42).

In Vitro Binding and Co-immunoprecipitation—A number of random mutations within the full-length Keap1 cDNA were generated using PCR and sequenced to identify the mutated codons. The cDNA encoding the Keap1-S383P protein contains a single T to C substitution at the first position in codon 383, resulting in a serine to proline substitution. The mutant cDNA was cloned into a CMV-based eukaryote expression vector. Expression vectors for the wild-type Keap1 and HA-tagged Nrf2 proteins have been described previously (21). The expression vectors were transfected into MDA-MB-231 cells and collected in radioimmune precipitation assay buffer (10 mM sodium phosphate pH 8.0, 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS) containing 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, and protease inhibitor mixture (Sigma). Immunoprecipitations were performed as described (21).

A portion of the Keap1-S383P cDNA encoding amino acids 321 to 609 was subcloned into pET15b and sequenced to confirm the presence of the S383P mutation. Wild-type and S383P Kelch domains containing an N-terminal hexahistidine tag and the Neh2 domain (amino acids 1–112), also containing an N-terminal hexahistidine tag, were purified using nitrilotriacetic acid affinity chromatography. The purified proteins were mixed together at a 1:1 molar ratio (100 pmol each) in a volume of 5 µl in 20 mM Tris-HCl, pH 7.5 containing 5 mM dithiothreitol at 4 °C for 2 h prior to electrophoresis (100 V) on a 7.5% polyacrylamide gel. The electrophoresis buffer was 5 mM Tris base, 40 mM glycine, pH 8.3.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Overall Structure—A recombinant protein consisting of the six kelch repeats of Keap1 (residues 321–609) was crystallized, and its structure determined at 1.85 Å resolution. Structure solution was performed using an SIRAS approach, utilizing the isomorphous and anomalous differences between native and SeMet crystals, as measured on a rotating anode x-ray system ({lambda} = 1.54 Å) (see "Experimental Procedures"). The current model extends from residue 322–609 of Keap1. The Kelch domain is a monomer in the crystal, consistent with characterization in solution by light scattering (data not shown). Although the Kelch domain has eight cysteine residues (Keap1 has 27 total), no disulfide bonds are found in the protein.

The six kelch repeats that comprise the Kelch domain of Keap1 form a highly symmetric, 6-bladed {beta}-propeller structure (Fig. 1, A and B), with approximate dimensions of 50 Å in diameter and 35 Å in height. A small channel (~6 Å diameter) runs through the center of the propeller, which is filled with a number of ordered solvent molecules. The Kelch domain has an overall negative charge (calculated pI = 6.5), but significant patches of positive charge are found on the surface of the protein, especially on the bottom side of the propeller (Fig. 1C).



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  		FIG. 1.
Structure of the Kelch domain of human Keap1. A, ribbon diagram of the Kelch domain (residues 322–609) shown in two orientations (top-down and side views) related by a 90° rotation. Each blade of the {beta}-propeller is shown in a different color and numbered I-VI. The four {beta}-strands found in each blade are labeled A–D (white font) on blade V. The N and C termini are both located in blade I and labeled N and C, respectively. Red arrow indicates the location of the S383P mutation that affects binding to Nrf2. B, topology diagram of the Kelch domain,illustrating the 4-stranded antiparallel {beta}-sheets in each blade, and the closure mechanism by a C-terminal strand in blade I. Residue numbers refer to the loops between strands; blades II and III have small regions of additional {beta}-sheet between strands B and C. Color scheme is the same as in A. C, electrostatic surface potential calculated by PYMOL (41) for the top and bottom surfaces of the Kelch domain. Red indicates negative charge; blue indicates positive. The top view of the electrostatic surface shows the protein in the same orientation as the top view in A.

 
As is typical of {beta}-propeller proteins, each blade of the Kelch domain is a twisted {beta}-sheet, composed of four antiparallel {beta}-strands (A–D). Strand A in each blade runs nearly parallel to the 6-fold axis of the propeller, and lines the central channel. Strands B–D exhibit progressively more twist and distance from the central axis, with strand D lying nearly perpendicular to the 6-fold and on the outside edge of the protein. The top of the propeller is formed by the loops between strands A and B, and strands C and D, whereas the bottom contains residues from the B-C loop and the interblade D-A loops. In each blade, the B-C loop extends beyond the D-A loop, which is essentially buried in the core of the protein. In the Kelch domain, blades II and III have additional ordered secondary structure in this loop, with small insertions of {beta}-sheet between strands B and C.

A key structural requirement of all {beta}-propeller proteins is the formation of a stable interface between their first and last blades, in this case between blades I and VI. In the Kelch domain {beta}-propeller, blades II through VI are formed by sequential stretches of residues. However, blade I contains three strands (B-D) from the N terminus of the protein, and one strand (A) from the C terminus. This arrangement, which effectively closes up the ring of blades in the propeller, is known as a C-terminal strand closure mechanism, and has been observed in other subclasses of {beta}-propellers (43). The seven blades of galactose oxidase, however, are closed via an N-terminal strand (44). Thus, the Kelch domain structure is the first example of a C-terminal closure mechanism for a member of the superfamily, and shows that kelch repeat proteins can utilize both types of closure mechanisms.

Structural Features of the Kelch Repeat—The kelch repeat is one of a number of different sequence motifs known to fold into {beta}-propellers, which also include the WD and YWTD repeats, and the aspartate box (43). Several highly conserved sequence features of the kelch motif distinguish it from other {beta}-propeller subtypes, including a glycine doublet following strand B, a tyrosine in strand C, and tryptophan in strand D (28). Several other positions in the motif have less conserved, but still notable sequence preferences, including four hydrophobic residues preceding the glycine doublet. Since the kelch repeat motif was identified after the structure determination of galactose oxidase (27), a detailed analysis of its structure with respect to sequence preferences has not yet been reported; a discussion of these features as observed in the Kelch domain of Keap1 follows.

Although the overall sequence identity between kelch repeats varies widely across the superfamily (28), the six blades of the Kelch domain are relatively similar in sequence and structure. A structure-based sequence alignment of the six kelch repeats from Keap1 is shown in Fig. 2A. The blades vary from 44 to 51 amino acids in length. Their pairwise sequence identities range from 29.5 to 48.9%, and the C{alpha} root-meansquare deviation (Rmsd) for the superpositions ranges from 0.53 to 1.0 Å2 (Fig. 2B). The largest variation in structure between the six blades is in the loops between the four {beta}-strands. In particular, additional residues between strands B and C of blade II extend this loop beyond that of the other blades at the bottom of the propeller. The overall structural similarity of the six blades observed in the Kelch domain corresponds to the previously noted higher degree of sequence similarity between kelch repeats within animal proteins as compared with those in fungi or poxviruses, and led to the suggestion that these proteins arose from a more recent evolutionary divergence in the family (27).



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  		FIG. 2.
Sequence alignment and structural superposition of the six Kelch domain propellers. A, structure-based sequence alignment of the six kelch repeat motifs from the Kelch domain of Keap1. The residue range for each blade is indicated at the left; the total number of residues in each blade is listed at the right. The four conserved {beta}-strands in each blade are indicated above the sequences by arrows. Residues conserved in all six blades are highlighted in green; two other residues that play conserved structural roles (see text) are highlighted in pink. A consensus sequence for all six blades is shown at the bottom. B, three-dimensional superposition of the six blades of the {beta}-propeller of the Kelch domain, showing their highly conserved tertiary structure. The four {beta}-strands are labeled A–D. Color scheme is the same as in Fig. 1A; the conserved Tyr (pink) and Trp (gray) side chains are shown as sticks. Structural alignments were done with TOP3D (54).

 
The superposition of the six blades of the Kelch {beta}-propeller reveals key structural roles for the highly conserved residues in the kelch motif. Seven residues are completely conserved in all six blades: two of these are the strand C Tyr, and strand D Trp that help define the kelch motif. As seen in Fig. 2B, the C{alpha} positions of these two residues are essentially identical in all six blades. Furthermore, their side chains adopt highly similar conformations and are in van der Waals contact with each other. The conserved Tyr/Trp pairs are found near the top of the propeller at the interface between two blades, and are part of a well-packed hydrophobic core. In all six blades, the side chain NE1 atom of the conserved Trp forms a hydrogen bond with a backbone carbonyl of a residue in the loop following strand D of the previous blade (Fig. 3, A and C), thereby linking one blade to the next. In four of the six blades, the side chain hydroxyl of the conserved Tyr makes a hydrogen bond with an Asn or Asp residue five positions later in the sequence. This residue, in turn, hydrogen bonds to the backbone amide of the same residue that the Trp side chain contacts. Although this interaction is not completely conserved (in blade V a water molecule replaces the Asn/Asp side chain, and in blade VI where the ring closes, it is absent), it appears to be an important part of an interblade hydrogen bond network. It appears the side chains of the highly conserved Tyr/Trp residues help to both form the hydrophobic core of the protein and are also intimately involved in linking the blades of the {beta}-propeller.



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  		FIG. 3.
Close-up views of the conserved sequence elements in the kelch repeat motif of Keap1. A, view of the structural roles of the conserved Tyr and Trp in strands C and D the Kelch domain. Blades I and II are used as an example to illustrate the packing of these two residues at the interface between blades, and their participation in an interblade hydrogen bond network. B, illustration of the structural role of the conserved glycine doublet (pink) found in all six {beta}-propeller blades of the Kelch domain. Blade II is used as an example to illustrate the intrablade hydrogen bonding network. C, Fobs electron density map (green) showing a close-up view of the conserved Trp-Tyr interaction in blade II. Map is contoured at 1.0 {sigma}; phases are following solvent flattening by RESOLVE (33).

 
Another well-conserved sequence feature of the kelch repeat is the glycine doublet, found immediately following strand B of each blade. The backbone atoms of these two residues exhibit conformations normally accessible only to glycine ({varphi}/{psi} angles of ~66/-150° for the first, and 100/-180° for the second). Other residues occupying these positions would be in a highly unfavorable region of the Ramachandran plot, explaining the sequence conservation of this feature. The backbone amide and carbonyl of both glycines in the doublet also participate in hydrogen bonds (Fig. 3B). Two of these are with backbone atoms in residues preceding strand C, while a third is with a residue in a neighboring blade (not shown). The carbonyl of the first glycine, however, does not make a backbone contact, but rather hydrogen bonds with an Arg side chain in each blade. This Arg is located several residues prior to strand A and is conserved in all six blades (Fig. 2A). The conserved Arg, in turn, interacts with a Glu/Asp in strand C of the same blade (Fig. 3B). The Gly-Arg-Asp/Glu interaction is found in all six blades; however, in blade VI, a water molecule mediates the Gly-Arg contact. This conserved interaction would appear to be part of an intrablade hydrogen-bonding network that contributes to folding of the individual kelch repeats.

Comparison with Related Structures—The only other protein of known structure containing kelch repeat motifs is the fungal enzyme galactose oxidase (26, 44). There are a number of important differences between galactose oxidase and the Kelch domain of Keap1, which share only 16% amino acid identity overall. First, as noted previously, the kelch repeats in galactose oxidase form a 7-bladed {beta}-propeller, which is closed by an N-terminal strand, as opposed to the C-terminal closure in the 6-bladed propeller of the Kelch domain. Second, the kelch repeats of galactose oxidase are much more divergent from the canonical kelch repeat motif, than those in the Kelch domain of Keap1. For example, only three of the galactose oxidase blades contain the conserved Trp in strand D (26). In addition, galactose oxidase has a number of insertions between blades (3 of 12 or more residues relative to the Kelch domain), and the individual blades are less similar to each other, in both sequence and structure (pairwise sequence identities range from 0 to 34%, and C{alpha} Rmsd from 0.58 to 2.33 Å2 for structural super-positions). Thus the galactose oxidase structure, while critical for determining the structural family of the kelch repeat motif, is of limited utility for modeling many members of this rapidly expanding protein family. In contrast, the Kelch domain of Keap1, which contains six structurally similar examples of the kelch repeat whose sequences closely match the canonical repeat, should provide a high quality model for other members of the family, particularly for BTB-Kelch and other Keap1 proteins (see following section).

When compared with other proteins in the Protein Data Bank, the Kelch domain is most similar in overall structure to 6-bladed {beta}-propellers of other subtypes. A DALI search (45) reveals significant structural similarity to a number of proteins of widely varied function, including nidogen (46) and the low density lipoprotein receptor (47), both members of the YWTD {beta}-propeller family. Based on the DALI scores, other propeller subtypes are also obvious structural relatives, such as siali-dase, which contains the aspartate box sequence motif (43). At present, these structural comparisons do not indicate a closer relationship between the Kelch domain and any one particular subtype of {beta}-propeller protein, supporting its designation as a unique subclass within the {beta}-propeller family.

Disruption of Keap1-Nrf2 Interactions by a Single Amino Acid Substitution Located within the B-C Loop of Blade II—The Kelch domain of Keap1 binds the Neh2 domain of Nrf2 (14). The structure of the Kelch domain of Keap1 reveals several potential protein-protein interaction surfaces, including the top, the sides of each blade, and the bottom surface. To provide further insight into the surface(s) of the Kelch domain that are important for binding to Nrf2, a number of randomly generated point mutations within the Kelch domain of Keap1 were screened for their ability to bind Nrf2. One single point mutation, consisting of a proline substitution for serine 383 in Keap1, was identified which markedly reduced the ability of the isolated Kelch domain to bind the Neh2 domain using an in vitro gel mobility shift assay (Fig. 4A). A mutant full-length Keap1 protein containing the S383P substitution was also markedly reduced in its ability to bind the full-length Nrf2 protein as assessed by co-immunoprecipitation from transfected MDA-MB-231 cells (Fig. 4B, top panel). As expected, the Keap1-S383P protein did not down-regulate steady-state levels of Nrf2 (Fig. 4B, middle panel) or inhibit Nrf2-dependent gene expression (data not shown). Serine 383 is located at the tip of the B-C loop in blade II, which extends out from the bottom of the Kelch structure (Fig. 1A), suggesting that the Neh2 domain of Nrf2 binds to the bottom surface of the Kelch domain.



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  		FIG. 4.
A single amino acid substitution disrupts the ability of Keap1 to bind Nrf2. A, 100 pmol of the Neh2 domain (lanes 1 and 4), the wild-type (lane 2), or S383P (lane 5) Kelch domains, or a mixture containing 100 pmol each of the Neh2 domain and either of the respective Kelch domains (lanes 3 and 6) were electrophoresed through a 7.5% polyacrylamide gel to resolve the free proteins and the Kelch-Neh2 complex (lane 3). The proteins were visualized with Coomassie Blue stain. B, expression vectors for HA-Nrf2 (lanes 1–8), wild-type Keap1 (lanes 3 and 4), Keap1-S383P (lanes 5 and 6), and Keap1-DKelch were transfected into MDA-MB-231 cells. The cells were either untreated (odd number lanes) or treated with MG132 (even numbered lanes) for 5 hours prior to collection of cell lysates. A fraction of each cell lysate was analyzed by immunoblot to measure steady-state levels of HA-Nrf2 (middle panel) and the Keap1 proteins (bottom panel). Equivalent amounts of each cell lysate were subjected immunoprecipitation with anti-Keap1 antibodies followed by immunoblot analysis with anti-HA antibodies (top panel).

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Implications for the BTB-Kelch Family—BTB-Kelch proteins are a widespread and functionally diverse class of proteins characterized by an N-terminal BTB domain, a conserved central linker domain, and a C-terminal Kelch repeat domain. The BTB domain is a protein-protein interaction motif of known structure that mediates dimerization (48). BTB-Kelch proteins are common in metazoan animals, from Caenorhabditis elegans to humans, and have also been reported in animal poxvirus genomes. The frequent association of these two domains on a single polypeptide chain is demonstrated by a recent bioinformatics analysis of the kelch repeat motif, which identified more than 70 kelch repeat proteins in the human genome: the BTB-Kelch domain architecture was found in nearly three-fourths of these proteins (49).

The crystal structure of the Kelch domain of Keap1 can be used as a three-dimensional model for this domain in other members of the BTB-Kelch family. To illustrate this, we high-light the six blades of the {beta}-propeller and four strands within each blade on a sequence alignment of various family members (Fig. 5). This alignment includes the Kelch domain of Keap1 from five different species, and also the human proteins Mayven and GAN1. Mayven is an actin-binding protein and the human homolog of the Drosophila Kelch protein (50); GAN1 is also a member of the BTB-Kelch family (23). Autosomal recessive mutations of GAN1 are responsible for giant axonal neuropathy, a devastating disease characterized by neurofilament accumulation and axonal distension, which results in sensori-motor defects and early death, typically by the age of thirty (23).



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  		FIG. 5.
Sequence alignment of the Kelch domain of five Keap1 proteins and two other members of the BTB-Kelch superfamily. Yellow highlighting indicates the four {beta}-strands (A–D) of each blade as seen in the crystal structure of human Keap1; the six blades of the propeller are indicated by brackets. The Keap1 protein sequences are from human, mouse, zebrafish, D. melanogaster, and Anopheles gambiae, and have NCBI accession numbers of Q14145 [GenBank] , A9Z2X8, Q8JIM0, Q8IGL4, and XP313412, respectively. The two other proteins are Mayven (O95198 [GenBank] ) and GAN1 (Q9H2C0). A consensus sequence for the five Keap1 proteins is shown below the Anopheles sequence: residues conserved in all five proteins are shown in capital letters; residues conserved in four of the five sequences in lower case. Gray highlighting indicates solvent-exposed residues. Highly conserved residues in the kelch repeat motif are indicated below the alignment by *; residues of GAN1 whose mutation are linked with giant axonal neuropathy are in bold type and underlined. Alignment was done by ClustalW (55).

 
Sequence identities between the human Kelch domain and the selected proteins in our alignment range from 95% (between human and mouse Keap1) to 25% (between the human Kelch domain and GAN1). Only minor gaps/insertions are found over the nearly 300 aligned residues, and these are localized to regions outside of the four strands of the {beta}-propeller. (This is generally true across the superfamily as well; see Ref. 49). In the sequences shown in Fig. 5, the largest insertions are in the B-C loop on the bottom side of the propeller, notably in blades I and V of GAN1. Furthermore, sequence similarities at the C termini of the predicted propellers (strand A of blade I) make it likely that all of these proteins will utilize a C-terminal strand closure mechanism. This agrees with secondary structure predictions, which show a preference for {beta}-strand in this region of most members of the superfamily (28).

The sequence alignment shows that residues of structural importance to the kelch repeat motif (as identified by superposition of the six blades; see Fig. 2) are highly conserved in BTB-Kelch family members. In particular, we find that residues not typically considered part of the kelch repeat consensus sequence, such as the conserved Arg preceding strand A and the Asp/Glu in strand C, are also highly conserved in other family members. Indeed, sequence preferences for additional residues within the kelch repeat motif were previously used to identify a subset of more closely related BTB-Kelch proteins, which includes Keap1, Mayven, and GAN1 (49). Correlation between these residues of structural importance in the six blades of the Kelch domain, with those of sequence conservation in this subset of the BTB-Kelch family, indicate that all of these proteins are likely to form highly symmetric 6-bladed {beta}-propellers, similar to the Kelch domain of human Keap1.

Most members of the BTB-Kelch family have not yet been functionally characterized, including GAN1. However, a number of single amino acid mutations of this protein are clearly associated with giant axonal neuropathy (23, 51, 52). Due to the relevance of these mutations to human disease, we provide a brief overview of their predicted structural context in the GAN1 {beta}-propeller (see Fig. 5 and Table II). A compilation of these mutations shows they are found throughout the sequence, in five of the six blades. Using the crystal structure of the Kelch domain as a model, we find that the mutated residues are generally located in the "body" of the {beta}-propeller, rather than on external loops. (Two exceptions to this are Glu486, which is located on a surface exposed B-C loop, and Arg269, which is found at the disordered N terminus of the domain.) More specifically, the mutations primarily involve residues in either the {beta}-strands or in the extended D-A loop, which connects adjacent blades and is the more highly buried loop on the bottom side of the propeller.


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  		TABLE II
Disease-associated mutations in Kelch domain of GAN1

 
The specific structural effects of the GAN1 mutations cannot be predicted a priori, however, some clues may be derived from the roles of these residues in the Kelch domain (Table II). Mutations to highly conserved residues in the kelch repeat motif, would likely affect either intra- or interblade interactions described previously. The Y299C mutant, for example, would be expected to change both the packing of the conserved Tyr/Trp pair in the protein core, and also result in loss of the conserved, interblade Tyr-Asn/Asp hydrogen bond (Fig. 3A). Similarly, Leu309 and Ile423, although not absolutely conserved residues in the family, both pack against a conserved Tyr/Trp pair in the interior of the protein (Fig. 3C). Two other mutations, G368R and C570Y, are located in strand A of blades I and I, respectively. Strand A of each blade lines the central channel of the {beta}-propeller, and residues in this strand tend to have small, apolar side chains (or to be glycine) (Fig. 2), permitting the blades to pack tightly around the central axis of the protein; the large side chains of the mutated residues could be easily envisioned to interfere with this. Thus, in general, the GAN1 mutations appear to affect residues that could interfere with folding of the blades and/or the assembly of blades into the {beta}-propeller structure. As more disease-related mutants of GAN1 are isolated, the structure of the Kelch domain will be critical for understanding their structural context, and will also aid in biochemical and functional characterization of this protein.

Role of the Kelch Domain in Binding to Protein Substrates— Keap1 is a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex (18–20). Keap1 recruits Nrf2 into a functional ubiquitin ligase complex, thereby regulating the activity of Nrf2 through degradation. While Keap1 is the only BTB-Kelch protein shown to function as a substrate adaptor protein, several of the more than 50 BTB-Kelch proteins that are encoded by the human genome have been demonstrated to associate with Cul3 through their N-terminal BTB domain (24). Although substrates for these other BTB-Kelch proteins have not yet been identified, it seems likely that these proteins, like Keap1, will utilize their Kelch domain to recruit specific protein substrates into an E3 ubiquitin ligase complex.

The structure of the Kelch domain of Keap1 reveals several features that may be important for substrate binding. First, as noted previously, the B-C loops on the bottom of the propeller extend out from the body of the protein and are highly solvent accessible. Sequence comparisons of the BTB-Kelch family show that the B-C loops are quite variable in both amino acid sequence and length, although specific patterns of length and sequence conservation can be detected by comparing various family members. For example, the B-C loop in blade II in all of the Keap1 proteins is slightly longer than the corresponding loop in Mayven and GAN1 (Fig. 5). The structure of the Kelch domain of Keap1 reveals that the extra residues in this loop form a short {beta}-sheet that extends out from the bottom of the structure, and a single amino acid substitution within this {beta}-sheet extension disrupts the ability of the Kelch domain to bind Nrf2 (Fig. 4). In contrast, the B-C loops in blades I and V of GAN1 are longer than the corresponding loops in Keap1 or Mayven, and a single amino acid mutation within the B-C loop of blade V has been identified in a patient with giant axonal neuropathy (23). Based on these observations, we suggest that the bottom face of the Kelch {beta}-propeller is a likely surface for protein-protein interactions between BTB-Kelch substrate adaptor proteins and their substrate proteins. A similar arrangement has been reported for the F-box substrate adaptor {beta}-TrCP, which contains a C-terminal WD40 {beta}-propeller domain (53). Furthermore, we suggest that amino acid sequence variation in the B-C loops between the different BTB-Kelch proteins will contribute to their ability to bind specific substrates.

Another intriguing feature of the Kelch structure of Keap1 with potential relevance to substrate binding is the positively charged region on the bottom face of the propeller (Fig. 1C). This positively charged region is primarily caused by the highly conserved Arg residue found prior to strand A of each blade (Fig. 2). As noted previously, this Arg residue helps defines a subset (class I) of the human BTB-Kelch proteins, including Keap1 and GAN (49). In contrast, the other major subset of BTB-Kelch proteins, class II, does not have a conserved Arg in this position. Thus, it is likely that the presence of a positively charged region at the bottom of the Kelch domain may be a conserved structural feature that helps define the two subclasses of human BTB-Kelch proteins. Furthermore, as Nrf2 contains an acidic sequence motif, ETGE, which is required for binding to Keap1 (15), we suggest that electrostatic interactions between the ETGE motif and the positive charged bottom surface of the Kelch domain will contribute to binding of Nrf2 and Keap1. Experiments to test these hypotheses are currently underway in our laboratory.


   FOOTNOTES
 
The atomic coordinates and structure factors (code 1U6D) have been deposited in the Protein Data Bank, Research Collaboratory for Structural Bioinformatics, Rutgers University, New Brunswick, NJ (http://www.rcsb.org/).

* This work was supported by the University of Missouri Molecular Biology Program, the University of Missouri Food for the 21st Century program, Research Grants GM59213 and P50 CA103130 from the National Institutes of Health, and by a grant from the University of Missouri Research Board. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 573-882-6072; Fax: 573-884-4812; E-mail: Beamerl{at}missouri.edu.

1 The abbreviations used are: ARE, antioxidant response elements; Rmsd, root mean square domain; BTB, broad-complex, tramtrack, and bric a brac. Back


   ACKNOWLEDGMENTS
 
We thank Jack Tanner for the generous use of his FPLC.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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