Human Mitochondrial ABC Transporter, ABCB10 (plate form)

Target ID:

ABCB10A

Aliases:

ABCB10EST20237M-ABC2MTABC2

Deposition Date:

Wednesday 30th November 2011

Families:

Membrane proteins

Structure type:

apo

Download Coordinates:

4AYW

Authors:

A.C.W.PIKE,C.A.SHINTRE,Q.LI,J.KIM,F.VON DELFT,N.BURGESS-BROWN,O.GILEADI,A.BULLOCK,C.H.ARROWSMITH,J.WEIGELT,C.BOUNTRA,A.M.EDWARDS,E.P.CARPENTER

Site:

Oxford

4AYW

tabs

Structure Details

ABCB10 is a human mitochondrial ATP-binding cassette (ABC) transporter which is highly overexpressed in erythroid cells, cells that are committed to becoming red blood cells [1], [2], [3]. ABCB10 is embedded in the inner membrane of mitochondria, and is thought to pump its substrate out of the mitochondrial matrix into the intermembrane space. Human mitochondria have four ABC transporters, whereas yeast cells have three, including MDL1, which is highly homologous to ABCB10. Over expression of MDL1 in yeast cells lacking the gene for another mitochondrial ABC transporter, atm1, rescued cells under conditions of oxidative stress [4]. In mammals ABCB10 is important for hemoglobin and red blood cell formation and for protection of mitochondria against oxidative stress[5], [6]. Several alternative functional roles have been suggested for ABCB10 and its homologue MDL1 including transport of heme biosynthesis precursors [7], stabilisation of the iron transporter SLC25A37 [8] and peptide transport [9], [10]. However to date no substrate has been confirmed biochemically and the debate on ABCB10 function continues [3].

Here we present the structure of ABCB10 in complex with the ATP analogue AMP-PNP to 3.3Å resolution. This structure represents a different crystal form from the previous two ABCB10 structures and the construct used for crystallisation harbours an ATPase inactivating point mutation (R691H).

Materials and Methods

ABCB10A (4AYW) Materials & Methods

Entry Clone Source: MGC

Entry Clone Accession: IMAGE: 6143235

SGC Construct ID: ABCB10A-c007

GI Number: 9961244

Vector: pFB-LIC-Bse. Details [PDF ]; Sequence [ FASTA ] or [ GenBank ]

DNA sequence:
ATGCGAGGCCCCCCTGCCTGGGCAGG
GGACGAGGCCTGGCGGCGCGGGCCGG
CGGCGCCTCCCGGGGACAAGGGGCGG
CTGCGCCCCGCAGCGGCCGGACTCCC
GGAGGCCCGGAAGCTCCTGGGGCTGG
CGTACCCTGAGCGCCGGAGGCTGGCA
GCTGCGGTTGGATTTCTCACGATGTC
CAGTGTTATCTCCATGTCTGCCCCTT
TCTTCCTGGGGAAGATCATTGATGTC
ATCTATACCAACCCCACTGTGGACTA
CAGCGACAACCTGACCCGCCTCTGCC
TAGGGCTCAGTGCCGTGTTTCTGTGT
GGTGCTGCCGCCAATGCCATTCGTGT
CTACCTCATGCAAACTTCAGGTCAGC
GCATTGTGAATAGGCTGAGAACTTCA
TTATTCTCCTCCATTCTGAGGCAGGA
GGTTGCTTTCTTTGACAAGACTCGCA
CAGGAGAATTGATTAACCGCCTCTCA
TCAGACACTGCACTCCTGGGGCGCTC
AGTGACTGAAAACCTCTCAGATGGGC
TCAGGGCCGGGGCCCAGGCTTCTGTA
GGCATCAGTATGATGTTTTTTGTCTC
ACCTAATCTGGCCACCTTTGTTTTGA
GCGTGGTGCCTCCAGTGTCAATCATT
GCTGTAATTTATGGGCGATATCTACG
GAAACTGACCAAAGTCACTCAGGATT
CCCTGGCACAAGCCACTCAGCTAGCT
GAGGAACGTATTGGAAATGTAAGAAC
TGTTCGAGCTTTTGGGAAAGAAATGA
CTGAAATCGAGAAATATGCCAGCAAA
GTGGACCATGTAATGCAGTTAGCAAG
GAAAGAGGCATTCGCCCGGGCTGGTT
TCTTTGGAGCAACTGGGCTCTCCGGA
AACCTGATCGTGCTTTCTGTCCTGTA
CAAAGGAGGGCTGCTGATGGGCAGTG
CCCACATGACCGTGGGTGAACTCTCT
TCCTTCCTAATGTATGCTTTCTGGGT
TGGAATAAGCATTGGAGGTCTGAGCT
CTTTCTACTCGGAGCTGATGAAAGGA
CTGGGTGCAGGGGGGCGCCTCTGGGA
GCTCCTGGAGAGAGAGCCCAAGCTGC
CTTTTAACGAGGGGGTCATCTTAAAT
GAGAAAAGCTTCCAGGGTGCTTTGGA
GTTTAAGAACGTGCATTTTGCCTATC
CAGCTCGCCCAGAGGTGCCCATATTT
CAGGATTTCAGCCTTTCCATTCCGTC
AGGATCTGTCACGGCACTGGTTGGCC
CAAGTGGTTCTGGCAAATCAACAGTG
CTTTCACTCCTGCTGAGGTTGTACGA
CCCTGCTTCTGGAACTATTAGTCTTG
ATGGCCATGACATCCGTCAGCTAAAC
CCAGTGTGGCTGAGATCCAAAATTGG
GACAGTGAGTCAGGAACCCATTTTGT
TTTCTTGCTCTATTGCTGAGAACATT
GCTTATGGTGCTGATGACCCTTCCTC
TGTGACCGCTGAGGAAATCCAGAGAG
TGGCTGAAGTGGCCAATGCAGTGGCC
TTCATCCGGAATTTCCCCCAAGGGTT
CAACACTGTGGTTGGAGAAAAGGGTG
TTCTCCTCTCAGGTGGGCAGAAACAG
CGGATTGCGATTGCCCGTGCTCTGCT
AAAGAATCCCAAAATTCTTCTCCTAG
ATGAAGCAACCAGTGCGCTGGATGCC
GAAAATGAGTACCTTGTTCAAGAAGC
TCTAGATCGACTGATGGATGGAAGAA
CGGTGTTAGTTATTGCCCATCATCTG
TCCACCATTAAGAATGCTAATATGGT
TGCTGTTCTTGACCAAGGAAAAATTA
CTGAATATGGAAAACATGAAGAGCTG
CTTTCAAAACCAAATGGGATATACAG
AAAACTAATGAACAAACAAAGTTTTA
TTTCAGCATGA

Expressed sequence (small letters refer to tag sequence):
mghhhhhhssgvdlgtenlyfqs^MR
GPPAWAGDEAWRRGPAAPPGDKGRLR
PAAAGLPEARKLLGLAYPERRRLAAA
VGFLTMSSVISMSAPFFLGKIIDVIY
TNPTVDYSDNLTRLCLGLSAVFLCGA
AANAIRVYLMQTSGQRIVNRLRTSLF
SSILRQEVAFFDKTRTGELINRLSSD
TALLGRSVTENLSDGLRAGAQASVGI
SM MFFVSPNLATFVLSVVPPVSIIA
VIYGRYLRKLTKVTQDSLAQATQLAE
ERIGNVRTVRAFGKEMTEIEKYASKV
DHVMQLARKEAFARAGFFGATGLSGN
LIVLSVLYKGGLLMGSAHMTVGELSS
FLMYAFWVGISIGGLSSFYSELMKGL
GAGGRLWELLEREPKLPFNEGVILNE
KSFQGALEFKNVHFAYPARPEVPIFQ
DFSLSIPSGSVTALVGPSGSGKSTVL
SLLLRLYDPASGTISLDGHDIRQLNP
VWLRSKIGTVSQEPILFSCSIAENIA
YGADDPSSVTAEEIQRVAEVANAVAF
IRNFPQGFNTVVGEKGVLLSGGQKQR
IAIARALLKNPKILLLDEATSALDAE
NEYLVQEALDRLMDGRTVLVIAHHLS
TIKNANMVAVLDQGKITEYGKHEELL
SKPNGIYRKLMNKQSFISA

Tags and additions: N-terminal, TEV cleavable hexahistidine tag. (^ cleavage site)

Host: Spodoptera frugiperda (SF9) insect cells

Growth Medium & Induction Protocol: Insect cells with a density of 2x10⁶ per litre of cell culture in SF900 medium (Invitrogen) were infected with recombinant baculovirus (5ml P2 virus per litre cell culture) and incubated for 72 hours. Cells were harvested by centrifugation and the pellet was flash frozen in liquid N₂.

Extraction buffer, extraction method: Frozen pellets were thawed and re-suspended in hypotonic buffer for lysis using a dounce homogeniser and DIAX homogeniser (Heidolph) at 10,000 r.p.m for 2 minutes. Cell membranes were collected by ultracentrifugation at 100,000Xg and the homogenisation repeated. The membranes were further washed with hypertonic buffer twice and the washed membranes were re-suspended in extraction buffer supplemented with 1% DDM, 0.1% CHS and incubated at 4°C with stirring. The extracted protein was separated from insoluble membranes by ultracentrifugation and collected for purification.

Hypotonic buffer: 10mM HEPES pH7.5, 0.5 mM EDTA

Hypertonic buffer: 10mM HEPES pH7.5,1M NaCl, 0.5mM EDTA

Extraction/wash buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl; 20 mM imidazole, 0.5mM MgCl₂ and 0.5 mM TCEP

Column 1: Co-affinity. Cobalt Talon (Clontech), 4 ml of 50 % slurry in 1.5 x 10 cm column, washed with extraction/ wash buffer.

Column 1 Buffers:
Extraction/wash buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl; 20 mM imidazole, 0.5mM MgCl2 and 0.5 mM TCEP ,0.02%DDM, 0.002%CHS
Elution buffer: 50 mM HEPES, pH 7.5; 200 mM NaCl; 300 mM imidazole, 0.5mM MgCl2 and 0.5 mM TCEP, 0.02%DDM, 0.002%CHS

Column 1 Procedure: The solubilised membrane protein was batch bound to Cobalt Talon resin equilibrated with extraction buffer at 4°C for one hour and subsequently poured into a gravity flow glass column. The column was then washed with 25CV of wash buffer followed by elution in 1CV fractions until all the protein was eluted.

Enzymatic Treatment: 1mg TEV protease was added overnight at 4°C to purified protein with concurrent buffer exchange by dialysis using a 3kDa cut off dialysis membrane into gel filtration buffer. The protease was removed and tag cleaved protein was collected by binding to Cobalt talon and collecting the flow-through.

Column 2: Size Exclusion Chromatography. Superose6 (GE healthcare)

Column 2 Buffer:
Dialysis/ Gel Filtration buffer: 20 mM HEPES pH 7.5, 200 mM NaCl, 0.5 mM TCEP, 0.5mM MgCl₂, 0.02% DDM+0.001%Cardiolipin

Column 2 Procedure: The protein was concentrated and applied to a Superose6 gel filtration column equilibrated with gel filtration buffer using an AKTA Prime system.

Mass spec characterization:
The purified protein was homogeneous and had an experimental mass of 67075 Da. This represents an unexplained, average mass of +91Da when compared to the calculated mass (66984 Da) based on the known sequence. 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% methanol in water with 0.1% formic acid.

Protein Concentration: Protein was concentrated to >8mg/ml using a 100kDa cut-off concentrator and back diluted to 5-8mg/ml with the addition of 2mM Mg-AMP-PNP.

Crystallization: Crystals were grown in sitting drops at 20°C.

Drops (200nl) comprising protein solution (5mg/ml; ABCB10A DDM/CHS/AMP-PNP) and reservoir solution (0.1-0.2 M NaCl, 5-7% (v/v) jeffamine M600, 30-40% (v/v) PEG400, 0.1 M glycine pH 9.5) in protein:reservoir ratios of 2:1 or 1.5:1 were equilibrated against 20µl of the same reservoir solution.

Crystal plates were transferred to 6°C prior to directly flash cooling crystals in liquid nitrogen.

Data Collection:
Resolution: 3.3Å
Data were collected at 100°K using a spiral scan collection strategy and a 10x50µm beamsize on I24 microfocus beamline (Diamond Light Source, UK).

Native data were collected to 3.4Å from a single crystal (Λ=0.9686Å). Data for a mercury derivative, prepared by soaking native crystals overnight with 1mM EMTS, were collected to 4Å (µ=0.9779Å). A higher resolution native dataset (nominally 3.15Å) was collected from a crystal soaked for 5mins in mother liquor containing 10mM lutetium chloride.

Structure Solution: Initial phases were calculated using SIRAS. Two Hg sites were located with SHELXD and phase refinement was carried out with SHARP / SOLVE. Phase extension / density modification to 3.4Å yielded an interpretable map and allowed an initial backbone trace to be built. However, both data anisotropy and disorder of the NBD hindered model completion using the plate form crystals. The structure was finally solved using the refined, higher resolution rod-form A structure. Molecular replacement was used to position the separate transmembrane and nucleotide-binding domains. Refinement was carried out with autoBUSTER using LSSR restraints to the higher resolution rod-form A structure.

References

1. Shirihai, O.S., et al., ABC-me: a novel mitochondrial transporter induced by GATA-1 during erythroid differentiation. EMBO J, 2000. 19(11): p. 2492-502.
Lill, R. and G. Kispal, Mitochondrial ABC transporters. Res Microbiol, 2001. 152(3-4): p. 331-40.
Zutz, A., et al., Mitochondrial ABC proteins in health and disease. Biochim Biophys Acta, 2009. 1787(6): p. 681-90.
Chloupkova, M., L.S. LeBard, and D.M. Koeller, MDL1 is a high copy suppressor of ATM1: evidence for a role in resistance to oxidative stress. J Mol Biol, 2003. 331(1): p. 155-65.
Hyde BB, Liesa M, Elorza AA, Qiu W, Haigh SE, Richey L, Mikkola HK, Schlaeger TM, Shirihai OS. The mitochondrial transporter ABC-me (ABCB10), a downstream target of GATA-1, is essential for erythropoiesis in vivo. Cell Death Differ. 2012 Jul;19(7):1117-26. doi: 10.1038/cdd.2011.195. Epub 2012 Jan 13.)
Liesa M, Luptak I, Qin F, Hyde BB, Sahin E, Siwik DA, Zhu Z, Pimentel DR, Xu XJ, Ruderman NB, Huffman KD, Doctrow SR, Richey L, Colucci WS, Shirihai OS. Mitochondrial transporter ATP binding cassette mitochondrial erythroid is a novel gene required for cardiac recovery after ischemia/reperfusion. Circulation. 2011 Aug 16;124(7):806-13. Epub 2011 Jul 25. PMID: 21788586
Chen, W., H.A. Dailey, and B.H. Paw, Ferrochelatase forms an oligomeric complex with mitoferrin-1 and Abcb10 for erythroid heme biosynthesis. Blood, 2010. 116(4): p. 628-30.
Chen, W., et al., Abcb10 physically interacts with mitoferrin-1 (Slc25a37) to enhance its stability and function in the erythroid mitochondria. Proc Natl Acad Sci U S A, 2009. 106(38): p. 16263-8.
Young, L., et al., Role of the ABC transporter Mdl1 in peptide export from mitochondria. Science, 2001. 291(5511): p. 2135-8.
Hofacker, M., et al., Structural and functional fingerprint of the mitochondrial ATP-binding cassette transporter Mdl1 from Saccharomyces cerevisiae. J Biol Chem, 2007. 282(6): p. 3951-61.