Human kelch-like 7

Target ID:

KLHL7B

Aliases:

KLHL6KLHL7SBBI26

Deposition Date:

Friday 31st July 2009

Families:

Ubiquitin Related BiologyUbiquitylation - E3 Ligases

Structure type:

apo

Download Coordinates:

3II7

Authors:

Chaikuad, A., Thangaratnarajah, C., Cooper, C.D.O., Ugochukwu, E., Muniz, J.R.C., Krojer, T., Sethi, R., Pike, A.C.W., Filippakopoulos, P., von Delft, F., Arrowsmith, C.H., Edwards, A.M., Weigelt, J., Bountra, C., Knapp, S., Bullock, A.

Site:

Oxford

3II7

tabs

Structure Details

Kelch-like 7 is a member of the BTB/kelch (Broad-Complex, Tramtrack and Bric a brac) repeat family. The protein consists of a BTB domain, a BACK domain (BTB and C-terminal Kelch), and a C-terminal Kelch domain comprising six Kelch repeats.

KLHL7 is ubiquitously expressed with highest expression levels in heart, nervous system and testis.

KLHL7 was originally identified as an autoantigen in several cancers, neurological diseases as well as Sjögren's syndrome (Bredholt et al., 2006), (Uchida et al., 2005). More recently it has been shown that mutations in the BACK domain of KLHL7 can lead to autosomal-dominant retinitis pigmentosa (Friedman et al., 2009). Although the precise function for KLHL7 is unknown it is thought by analogy with other family members to function as an E3 ubiquitin ligase in which the Kelch domain forms the substrate recognition unit, while the BTB domain complexes with Cullin3; the BACK domain is thought to represent a helical linker.

Here we report the structure of the Kelch domain of KLHL7 at 1.6 Å resolution.

Materials and Methods

Entry Clone Source: MGC

Entry Clone Accession: BC039585

SGC Construct ID: KLHL7B-c009

GenBank GI number: gi|170784846

Vector: pNIC28-Bsa4. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

Coding DNA sequence:
ATGGCAGCCTCTGGGGTGGAGAAGAGCAGC
AAGAAGAAGACCGAGAAGAAACTTGCTGCT
CGGGAAGAAGCTAAATTGTTGGCGGGTTTC
ATGGGCGTCATGAATAACATGCGGAAACAG
AAAACGTTGTGTGACGTGATCCTCATGGTC
CAGGAAAGAAAGATACCTGCTCATCGTGTT
GTTCTTGCTGCAGCCAGTCATTTTTTTAAC
TTAATGTTCACAACTAACATGCTTGAATCA
AAGTCCTTTGAACTAGAACTCAAAGATGCT
GAACCTGATATTATTGAACAACTGGTGGAA
TTTGCTTATACTGCTAGAATTTCCGTGAAT
AGCAACAATGTTCAGTCTTTGTTGGATGCA
GCAAACCAATATCAGATTGAACCTGTGAAG
AAAATGTGTGTTGATTTTTTGAAAGAACAA
GTTGATGCTTCAAATTGTCTTGGTATAAGT
GTGCTAGCGGAGTGTCTAGATTGTCCTGAA
TTGAAAGCAACTGCAGATGACTTTATTCAT
CAGCACTTTACTGAAGTTTACAAAACTGAT
GAATTTCTTCAACTTGATGTCAAGCGAGTA
ACACATCTTCTCAACCAGGACACTCTGACT
GTGAGAGCAGAGGATCAGGTTTATGATGCT
GCAGTCAGGTGGTTGAAATACGATGAACCT
AATCGCCAGCCATTTATGGTTGATATCCTT
GCTAAAGTCAGGTTTCCTCTTATATCAAAG
AATTTCTTAAGTAAAACGGTACAAGCTGAA
CCACTTATTCAAGACAATCCTGAATGCCTT
AAGATGGTGATAAGTGGAATGAGGTACCAT
CTACTGTCTCCAGAGGACCGAGAAGAACTT
GTAGATGGCACAAGACCTAGAAGAAAGAAA
CATGACTACCGCATAGCCCTATTTGGAGGC
TCTCAACCACAGTCTTGTAGATATTTTAAC
CCAAAGGATTATAGCTGGACAGACATCCGC
TGCCCCTTTGAAAAACGAAGAGATGCAGCA
TGCGTGTTTTGGGACAATGTAGTATACATT
TTGGGAGGCTCTCAGCTTTTCCCAATAAAG
CGAATGGACTGCTATAATGTAGTGAAGGAT
AGCTGGTATTCGAAACTGGGTCCTCCGACA
CCTCGAGACAGCCTTGCTGCATGTGCTGCA
GAAGGCAAAATTTATACATCTGGAGGTTCA
GAAGTAGGAAACTCAGCTCTGTATTTATTT
GAGTGCTATGATACGAGAACTGAAAGCTGG
CACACAAAGCCCAGCATGCTGACCCAGCGC
TGCAGCCATGGGATGGTGGAAGCCAATGGC
CTAATCTATGTTTGTGGTGGAAGTTTAGGA
AACAATGTTTCTGGGAGAGTGCTTAATTCC
TGTGAAGTTTATGATCCTGCCACAGAAACA
TGGACTGAGCTGTGTCCAATGATTGAAGCC
AGGAAGAATCATGGGCTGGTATTTGTAAAA
GACAAGATATTTGCTGTGGGTGGTCAGAAT
GGTTTAGGTGGTCTGGACAATGTGGAATAT
TACGATATTAAGTTGAACGAATGGAAGATG
GTCTCACCAATGCCATGGAAGGGTGTAACA
GTGAAATGTGCAGCAGTTGGCTCTATAGTT
TATGTCTTGGCTGGTTTTCAGGGTGTTGGT
CGATTAGGACACATTCTCGAATATAATACC
GAAACAGACAAATGGGTTGCCAACTCCAAA
GTTCGTGCTTTTCCAGTCACAAGTTGTTTA
ATTTGTGTTGTCGATACTTGTGGAGCAAAT
GAAGAGACCCTTGAAACATGAAAAATGAGT
GGACTTCAGACTCATCAGAGACTCTAA

Tags and additions: MHHHHHHSSGVDLGTENLYFQ*S(M), TEV-cleavable (*) N-terminal hexahistidine tag.

Expressed sequence (tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfqsmGTRPRRK
KHDYRIALFGGSQPQSCRYFNPKDYSWTDI
RCPFEKRRDAACVFWDNVVYILGGSQLFPI
KRMDCYNVVKDSWYSKLGPPTPRDSLAACA
AEGKIYTSGGSEVGNSALYLFECYDTRTES
WHTKPSMLTQRCSHGMVEANGLIYVCGGSL
GNNVSGRVLNSCEVYDPATETWTELCPMIE
ARKNHGLVFVKDKIFAVGGQNGLGGLDNVE
YYDIKLNEWKMVSPMPWKGVTVKCAAVGSI
VYVLAGFQGVGRLGHILEYNTETDKWVANS
KVRAFPVTSCLICVVDTCGANEETLET

Host: BL21 (DE3)R3-pRARE2 (Phage resistant strain)

Growth medium, induction protocol: A glycerol stock was used to inoculate a 50 ml starter culture containing LB media and 34 µg/ml chloramphenicol and 50 µg/ml kanamycin. The starter culture was grown overnight at 37°C with shaking at 250 rpm. The following morning three flasks containing 1 L LB media with 34 µg/ml chloramphenicol and 50 µg/ml kanamycin were each inoculated with 10 ml of the starter culture. Cultures were incubated at 37°C with shaking at 180 rpm until an OD₆₀₀ ˜0.5 was reached. The flasks were then cooled down to 18°C and 0.5 mM IPTG added to induce protein expression overnight. Cells were harvested by centrifugation at 4500 rpm at 4°C for 15 min. Cell pellets from each flask were resuspended in 30 ml binding buffer (50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole), transferred to 50 ml tubes, and stored at -20°C.

Extraction buffer, extraction method: The frozen cells were thawed and 0.5 mM TCEP, and 1 mM PMSF were added to the cell suspension. The cells were lysed by ultrasonication over 10 min with the sonicator pulsing ON for 5 sec and OFF for 15 sec. The cell lysate was spun down by centrifugation at 17000 rpm and 4°C for 1 h. The supernatant was recovered for purification.

Column 1: Anion-exchange for Nucleic acid removal with DEAE cellulose (DE52, Whatmann)
10 g of resin was suspended in 100 ml 2.5 M NaCl, and then applied onto a 2.5 x 20 cm column. The resin was then equilibrated with 100 ml binding buffer prior to loading the sample.

Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole; Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 30 mM imidazole

Procedure: The supernatant was first applied onto the column by gravity flow, which was followed by a wash with 100 ml wash buffer. The column flow-through and wash was directly applied onto a Ni-IDA column.

Column 2: Ni-Affinity Chromatography
5 ml of 50 % Ni-IDA slurry (Genscript) was applied onto a 1.5 x 10 cm column. The column was first washed with deionised destilled H2O, and then equilibrated with binding buffer.

Buffers: Binding buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 5 mM imidazole; Wash buffer: 50 mM Hepes, pH 7.5; 500 mM NaCl; 5% Glycerol; 30 mM imidazole; Elution buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 5% Glycerol; 50 to 250 mM imidazole

Procedure: The flow-through from column 1 (DE52) was applied by gravity flow onto the Ni-IDA column. The bound protein was eluted by applying a step gradient of imidazole using 7 ml portions of elution buffer with increasing concentration of imidazole (50 mM, 100 mM, 150 mM, 250 mM). 10 mM DTT was added to each fraction collected for overnight storage at 4°C.

Enzymatic treatment: TEV protease cleavage. Fractions containing KLHL7B were treated with TEV protease overnight at 4°C.

Column 3: Size Exclusion Chromatography S200 HiLoad 16/60 Superdex run on AKTA-Express

Buffer: Gel Filtration buffer: 300 mM NaCl, 50 mM Hepes pH 7.5, 0.5 mM TCEP

Procedure: Prior to applying the protein, the S200 16/60 column was washed and equilibrated with gel filtration buffer. Eluted protein from the Ni-IDA column was pooled and concentrated to 3 ml using an Amicon Ultra-15 filter with a 30 kDa cut-off. The concentrated protein was directly applied onto the equilibrated S200 16/60 column, and run at a flow-rate of 1 ml/min. The protein was eluted at 90 - 110 ml. Fractions containing the protein were pooled together, and 10 mM DTT was added for overnight storage at 4°C.

Column 4: Cation-exchange - MonoS 5/50 GL column on AKTA-Purifier

Buffers: Buffer A (low salt): 50 mM Hepes pH 7.5; Buffer B (high salt): 50 mM Hepes, pH 7.5; 1 M NaCl

Procedure: Prior to applying the protein, the MonoS column was washed with buffer B before equilibration with buffer A. The eluted protein from gel filtration was diluted with buffer A to 50 ml then applied to a MonoS column, and run at a flow-rate of 1 ml/min with a linear gradient. The protein was eluted at 9.8 % buffer B. Fractions containing the protein were pooled and 10 mM DTT was added. Protein was stored at 4°C.

Concentration: The protein was concentrated in an Amicon Ultra-4 filter with a 10 kDa cut-off.

Mass spectrometry characterization : The purified protein was homogeneous and had an experimental mass of 34.039 kDa, as expected from its primary structure. Masses were determined by LC-MS, using an Agilent LC/MSD TOF system with reversed-phase HPLC coupled to electrospray ionisation and an orthogonal time-of-flight mass analyser. Proteins were desalted prior to mass spectrometry by rapid elution off a C3 column with a gradient of 5-95 % acetonitrile in water with 0.1 % formic acid.

Crystallization: Protein was buffered in 50 mM HEPES, pH 7.4, 100 mM NaCl, 10 mM DTT. Protein was concentrated down to 10.10 mg/ml (calculated using an extinction coefficient of 66350). Crystals were grown at 20°C in 150 nl sitting drops mixing 50 nl protein solution with 100 nl of a reservoir solution containing 0.1M MES, pH 6.5, 12% PEG 20K. On mounting crystals were cryo-protected with an additional 30% ethylene glycol.

Data Collection: Resolution: 1.6Å, X-ray source: Rigaku FR-E Superbright

References

Bredholt, G., Storstein, A., Haugen, M., Krossnes, B.K., Husebye, E., Knappskog, P., and Vedeler, C.A. (2006). Detection of autoantibodies to the BTB-kelch protein KLHL7 in cancer sera. Scand J Immunol 64, 325-335.

Friedman, J.S., Ray, J.W., Waseem, N., Johnson, K., Brooks, M.J., Hugosson, T., Breuer, D., Branham, K.E., Krauth, D.S., Bowne, S.J., et al. (2009). Mutations in a BTB-Kelch protein, KLHL7, cause autosomal-dominant retinitis pigmentosa. Am J Hum Genet 84, 792-800.

Uchida, K., Akita, Y., Matsuo, K., Fujiwara, S., Nakagawa, A., Kazaoka, Y., Hachiya, H., Naganawa, Y., Oh-Iwa, I., Ohura, K., et al. (2005). Identification of specific autoantigens in Sjögren's syndrome by SEREX. Immunology 116, 53-63.