Human glycogen branching enzyme (GBE1)

Target ID:

GBE1A

Aliases:

GBEGBE1

Deposition Date:

Wednesday 31st July 2013

Families:

Miscellaneous

Structure type:

apo

Download Coordinates:

4BZY

Authors:

D.S.Froese, T.Krojer, S.Goubin, C.Strain-Damerell, P.Mahajan, F.von Delft, N.Burgess-Brown, C.Bountra, C.H.Arrowsmith, A.Edwards, W.W.Yue

Site:

Oxford

4BZY

tabs

Structure Details

Glycogen is a compact polymer of α 1,4-linked glucose units regularly branched with α-1,6-glucosidic bonds, serving as the main carbohydrate store and energy reserve in bacterial and animal kingdoms. In eukaryotes, glycogenin initiates the synthesis of the glucan chain, which is further elongated by glycogen synthase. In the final step, side chains are then introduced by glycogen branching enzyme (GBE) which catalyses the formation of α-1,6-linked branch points. The branched glycogen structure, occurring every 8-14 glucose residues, increases the number of reactive termini, and also increases polymer solubility by reducing the osmotic pressure within cells [1].

Inherited mutations in the human gbe1 gene (chromosome 3p12.3) cause the glycogen storage disorder type IV (OMIM 232500) [2, 3], an autosomal recessive disorder characterized by the deposit of glycogen with fewer branch points and longer outer chains. This abnormal glycogen forms an insoluble structure (polyglucosan) that resembles amylopectin in affected tissues e.g. liver, heart, muscle, nervous system and skin. A majority of disease causing mutations are of the missense type, likely to affect the GBE protein structure and function.

At the protein level, GBE catalyzes two steps within the same active site: first it cleaves an α-1,4-linked segment (> 6 glucosyl units) from the outer non-reducing end of a glucan chain (amylase-type hydrolysis). Second, it transfers the cleaved oligosacchride, via an α-1,6 bond, to the primary C6 hydroxyl of a glucose unit in the same or neighbouring chain (transglycoslation/isomerization). Until now, structural information of GBE is available from plant and bacteria, but not in mammals. To this end, we report the structure determination of human GBE1.

Materials and Methods

Target ID: GBE1A
Entry Clone ID: GBE1A-s001
Allele ID: GBE1A-a106
Construct ID GBE1A-c106
Clone ID GBE1A-k106
Expression ID GBE1A-e118
Purification ID: GBE1A-p004
Entry clone source: MGC
Entry clone accession: gi| 4574938
Vector: pFB-LIC-Bse
Cell line: DH10Bac
Tags and additions: N-terminal, TEV protease cleavable hexahistidine tag

Coding DNA sequence

CCATGGGCCACCATCATCATCATCAT
TCTTCTGGTGTAGATCTGGGTACCGA
GAACCTGTACTTCCAATCCATGTTGA
AGCCCTACGCCGTGGACTTCCAGCGC
AGGTATAAGCAGTTTAGCCAAATTTT
GAAGAACATTGGAGAAAATGAAGGTG
GTATTGATAAGTTTTCCAGAGGCTAT
GAATCATTTGGCGTCCACAGATGTGC
TGATGGTGGTTTATACTGCAAAGAAT
GGGCCCCGGGAGCAGAAGGAGTTTTT
CTTACTGGAGATTTTAATGGTTGGAA
TCCATTTTCGTACCCATACAAAAAAC
TGGATTATGGAAAATGGGAGCTGTAT
ATCCCACCAAAGCAGAATAAATCTGT
ACTCGTGCCTCATGGATCCAAATTAA
AGGTAGTTATTACTAGTAAAAGCGGA
GAGATCTTGTATCGTATTTCACCGTG
GGCAAAGTATGTGGTTCGTGAAGGTG
ATAATGTGAATTATGATTGGATACAC
TGGGATCCAGAACACTCATATGAGTT
TAAGCATTCCAGACCAAAGAAGCCAC
GGAGTCTAAGAATTTATGAATCTCAT
GTGGGAATTTCTTCCCATGAAGGAAA
AGTAGCTTCTTATAAACATTTTACAT
GCAATGTACTACCAAGAATCAAAGGC
CTTGGATACAACTGCATTCAGTTGAT
GGCAATCATGGAGCATGCTTACTATG
CCAGCTTTGGTTACCAAATCACAAGC
TTCTTTGCAGCTTCCAGCCGTTATGG
ATCACCTGAAGAGCTACAAGAACTGG
TAGACACAGCTCATTCCATGGGTATC
ATAGTCCTCTTAGATGTGGTACACAG
CCATGCTTCAAAAAATTCAGCAGATG
GATTGAATATGTTTGATGGGACAGAT
TCCTGTTATTTTCATTCTGGACCTAG
AGGGACTCATGATCTTTGGGATAGCA
GATTGTTTGCCTACTCCAGCTGGGAA
GTTTTAAGATTCCTTCTGTCAAACAT
AAGATGGTGGTTGGAAGAATATCGCT
TTGATGGATTTCGTTTTGATGGTGTT
ACGTCCATGCTTTATCATCACCATGG
AGTGGGTCAAGGTTTCTCAGGTGATT
ACAGTGAATATTTCGGACTACAAGTA
GATGAAGATGCCTTGACTTACCTCAT
GTTGGCAAATCATTTGGTTCACACGC
TGTGTCCCGATTCTATAACAATAGCT
GAGGATGTATCAGGAATGCCAGCTCT
GTGCTCTCCAATTTCCCAGGGAGGGG
GTGGTTTTGACTATCGACTAGCCATG
GCAATTCCAGATAAGTGGATTCAGCT
ACTTAAAGAGTTTAAAGATGAAGACT
GGAACATGGGCGATATAGTATACACG
CTCACAAACAGGCGCTACCTTGAAAA
GTGCATTGCTTATGCAGAGAGCCATG
ATCAGGCATTGGTTGGGGATAAGTCG
CTGGCATTTTGGTTGATGGATGCCGA
AATGTATACAAACATGAGTGTCCTGA
CTCCTTTTACTCCAGTTATTGATCGT
GGAATACAGCTTCATAAAATGATTCG
ACTCATTACGCATGGGCTTGGTGGAG
AAGGCTATCTCAATTTCATGGGTAAT
GAATTTGGGCATCCTGAATGGTTAGA
CTTCCCAAGAAAAGGAAATAATGAGA
GTTACCATTATGCCAGGCGGCAGTTT
CATTTAACTGACGACGACCTTCTTCG
CTACAAGTTCCTAAATAATTTTGACA
GGGATATGAATAGATTGGAAGAAAGA
TATGGTTGGCTTGCAGCTCCACAGGC
CTACGTGAGTGAAAAACATGAAGGCA
ATAAGATCATTGCTTTTGAAAGAGCA
GGTCTTCTTTTCATTTTCAACTTCCA
TCCAAGCAAGAGCTACACTGACTACC
GAGTTGGAACAGCATTGCCAGGGAAA
TTCAAAATTGTGCTAGATTCAGATGC
AGCGGAATATGGAGGGCATCAGAGAC
TGGACCACAGCACTGACTTTTTTTCT
GAGGCTTTTGAACATAATGGGCGTCC
CTATTCTCTTTTGGTGTACATTCCAA
GCAGAGTGGCCCTCATCCTTCAGAAT
GTGGATCTGTGACAGTAAAGGTGGAT
ACGGATCCGAATTCGAGCTCCGTCGA
CAAGCTT

Final protein sequence

mhhhhhhssgvdlgtenlyfq*smLK
PYAVDFQRRYKQFSQILKNIGENEGG
IDKFSRGYESFGVHRCADGGLYCKEW
APGAEGVFLTGDFNGWNPFSYPYKKL
DYGKWELYIPPKQNKSVLVPHGSKLK
VVITSKSGEILYRISPWAKYVVREGD
NVNYDWIHWDPEHSYEFKHSRPKKPR
SLRIYESHVGISSHEGKVASYKHFTC
NVLPRIKGLGYNCIQLMAIMEHAYYA
SFGYQITSFFAASSRYGSPEELQELV
DTAHSMGIIVLLDVVHSHASKNSADG
LNMFDGTDSCYFHSGPRGTHDLWDSR
LFAYSSWEVLRFLLSNIRWWLEEYRF
DGFRFDGVTSMLYHHHGVGQGFSGDY
SEYFGLQVDEDALTYLMLANHLVHTL
CPDSITIAEDVSGMPALCSPISQGGG
GFDYRLAMAIPDKWIQLLKEFKDEDW
NMGDIVYTLTNRRYLEKCIAYAESHD
QALVGDKSLAFWLMDAEMYTNMSVLT
PFTPVIDRGIQLHKMIRLITHGLGGE
GYLNFMGNEFGHPEWLDFPRKGNNES
YHYARRQFHLTDDDLLRYKFLNNFDR
DMNRLEERYGWLAAPQAYVSEKHEGN
KIIAFERAGLLFIFNFHPSKSYTDYR
VGTALPGKFKIVLDSDAAEYGGHQRL
DHSTDFFSEAFEHNGRPYSLLVYIPS
RVALILQNVDL

Sequence MHHHHHHSSGVDLGTENLYFQ*SM is the purification tag plus TEV protease recognition site (*) (lower case in sequence).

Expression

High five cells were grown in Sf9 medium at 27°C. Cells were infected at a density of 2x106/ml with recombinant baculovirus (virus stock P2; 1ml of virus stock/1l of cell culture) Culture was supplemented with FCS to final concentration 1%. 120 hours post infection the cultures were collected and centrifuged for 20min at 1500 xg. Cellular pellets were discarded and supernatants were used as a source of a protein.

Purification

Cell Lysis

Cell pellets were dissolved in approximately 50ml lysis buffer and broken by sonication for 10min @ 35% amplitude followed by homogenization by 2 passes at 12,000 psi. The cell debris was pelleted at 40,000 x g and the supernatant used for further purification.

Lysis Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole pH 7.4, 0.5 mM TCEP, 1 tablet per 50 ml protease inhibitor cocktail EDTA-free (Roche)

Column 1: Ni-NTA (1.5 ml volume in a gravity-flow column).

The clarified cell extract was added to 1.5 ml of Ni-NTA pre-equilibrated with lysis buffer and passed through a glass column. The column was then washed with Binding Buffer (2 x 15 ml) and Wash Buffer (2 x 15 ml). The protein was eluted with 25 ml of Elution Buffer in 5 x 2.5 ml fractions.

Binding Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 10 mM Imidazole pH 7.4, 0.5 mM TCEP

Wash Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 40 mM Imidazole pH 7.4, 0.5 mM TCEP

Elution Buffer: 50 mM Hepes pH 7.4, 500 mM NaCl, 5% Glycerol, 250 mM Imidazole pH 7.4, 0.5 mM TCEP

Column 2: Superdex 200 16/60 Gel Filtration.

The first three elution fractions from column 1 were pooled and concentrated to 5 ml with a 30 kDa mwco spin concentrator and injected into an s200 16/60 column (preequilibrated in GF Buffer) at 1.0 ml/min. 1.0 ml fractions were collected.

The protein eluted at between 80 ml and 90 ml volume.

GF Buffer: 10 mM Hepes pH 7.4, 500 mM NaCl, 0.5 mM TCEP, 5% Glycerol.

Concentration

Pooled protein fractions were concentrated to 16.5 mg/ml using a 30 kDa mwco concentrator.

Mass spectrometry characterisation

After TEV protease digestion:

Measured mass: 78939.2 Da

Expected mass: 79023.3 Da

Crystallization

Crystals were grown by vapour diffusion in sitting drop at 4°C. A sitting drop consisting of 50 nl protein and 100 nl well solution was equilibrated against well solution containing 0.05 M sodium-succinate and 22% PEG 3350 supplemented with 4% formamide (Hampton additive screen). Crystals were mounted in the presence of 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen.

Data collection

Resolution: 2.70 Å

X-ray source: Diamond I04

References

Thon, V.J., M. Khalil, and J.F. Cannon, Isolation of human glycogen branching enzyme cDNAs by screening complementation in yeast. J Biol Chem, 1993. 268(10): p. 7509-13.
Magoulas, P.L., et al., Diffuse reticuloendothelial system involvement in type IV glycogen storage disease with a novel GBE1 mutation: a case report and review. Hum Pathol, 2012. 43(6): p. 943-51.
Moses, S.W. and R. Parvari, The variable presentations of glycogen storage disease type IV: a review of clinical, enzymatic and molecular studies. Curr Mol Med, 2002. 2(2): p. 177-88.