Human Glycogenin-1 in complex with manganese and UDP, in a triclinic closed form

Target ID:

GYG1A

Deposition Date:

Saturday 30th July 2011

Families:

Miscellaneous

Structure type:

protein-ligand

Download Coordinates:

3T7M

Authors:

Chaikuad, A., Froese, D.S., Krysztofinska, E., von Delft, F., Weigelt, J., Arrowsmith, C.H., Edwards, A.M., Bountra, C., Oppermann, U., Yue, W. W

Site:

Oxford

ICM Datapack:

ICM Datapack

3T7M

tabs

Structure Details

Glycogen is a polymer of α-1,4-linked glucose branched by α-1,6-glycosidic linkages, and serves as a major storage reserve of glucose for organisms from bacteria to human. The biosynthesis of glycogen is initiated by the priming protein glycogenin (EC 2.4.1.186) [1], which synthesizes a linear α-1,4-linked glucan chain of up to ~10 glucose units attached to a protein tyrosyl residue. Subsequently, the major biosynthesis enzyme glycogen synthase will take over the bulk elongation of the glucan chain, in concert with the branching enzyme that catalyzes the α-1,6-glycosidic bond formation every 10-13 glucose units. In mammals there are two glycogenin isoforms, namely muscle GYG1 and liver GYG2 [2], sharing in common catalytic core and GS binding site (~350 aa), while GYG2 contains a unique ~110 aa extension of unknown function at the C-terminus. GYG1/GYG2 catalyzes a self-glucosylation reaction using UDP-glucose as sugar donor, and as sugar acceptor a tyrosine residue (in the first step) or the C4-hydroxyl of the terminal glucose unit from the nascent glucan chain (in subsequent steps). The disease relevance of glycogenin is highlighted by the recent identification of patient mutations in the human gyg1 gene (e.g. missense variant Thr83Met) that caused muscle weakness and cardiac arrhythmia, linking glycogenin deficiency to a new form of glycogen storage disorder [3]. Here we present the crystal structure of human GYG1 bound with Mn2+-UDP in the triclinic (PDB code 3T7M) and monoclinic (PDB code 3T7N) crystal form, as well as GYG1 bound with Mn2+-UDP-glucose (PDB code 3T7O). A comparison of these structures reveals dramatic ligand-induced conformational changes in three mobile regions of the protein.

Materials and Methods

Entry Clone Source: IMAGE

Entry Clone Accession: IMAGE:3504538

SGC Construct ID: GYG1A-c003

GenBank GI number: gi|20127457

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

Amplified construct sequence:
CATATGCACCATCATCATCATCATTC
TTCTGGTGTAGATCTGGGTACCGAGA
ACCTGTACTTCCAATCCATGACAGAT
CAGGCCTTTGTGACACTAACCACAAA
CGATGCCTACGCCAAAGGTGCCCTGG
TCCTGGGATCATCTCTGAAACAGCAC
AGGACCACCAGGAGGCTGGTCGTGCT
CGCCACCCCTCAGGTCTCAGACTCCA
TGAGAAAAGTTTTAGAGACAGTCTTT
GATGAAGTCATCATGGTAGATGTCTT
GGACAGTGGCGATTCTGCTCATCTAA
CCTTAATGAAGAGGCCAGAGTTGGGT
GTCACGCTGACAAAGCTCCACTGCTG
GTCGCTTACACAGTATTCAAAATGTG
TATTCATGGATGCAGATACTCTGGTC
CTAGCAAATATTGATGATCTTTTTGA
CAGAGAAGAATTGTCAGCAGCACCAG
ACCCAGGGTGGCCTGACTGCTTCAAT
TCCGGAGTCTTCGTTTATCAGCCTTC
AGTTGAAACATACAATCAGCTGTTGC
ATCTTGCTTCTGAGCAAGGTAGTTTT
GATGGTGGGGACCAAGGCATACTGAA
CACATTTTTTAGCAGCTGGGCAACAA
CAGATATCAGAAAACACCTGCCGTTT
ATTTATAACCTAAGCAGCATCTCTAT
ATACTCCTACCTCCCGGCATTTAAAG
TGTTTGGTGCAAGTGCCAAAGTTGTG
CATTTCCTGGGACGAGTCAAACCATG
GAATTATACTTATGATCCCAAAACAA
AAAGTGTCAAAAGTGAGGCCCATGAT
CCCAACATGACTCATCCAGAGTTTCT
CATCCTGTGGTGGAACATCTTTACCA
CCAACGTTTTACCTCTGCTTCAATGA
CAGTAAAGGTGGATACGGATCCGAA

Final protein sequence (Tag sequence in lowercase):
mhhhhhhssgvdlgtenlyfq^smTD
QAFVTLTTNDAYAKGALVLGSSLKQH
RTTRRLVVLATPQVSDSMRKVLETVF
DEVIMVDVLDSGDSAHLTLMKRPELG
VTLTKLHCWSLTQYSKCVFMDADTLV
LANIDDLFDREELSAAPDPGWPDCFN
SGVFVYQPSVETYNQLLHLASEQGSF
DGGDQGILNTFFSSWATTDIRKHLPF
IYNLSSISIYSYLPAFKVFGASAKVV
HFLGRVKPWNYTYDPKTKSVKSEAHD
PNMTHPEFLILWWNIFTTNVLPLLQ

^ TEV cleavage site

Tags and additions: Cleavable N-terminal His₆ tag.

Host: E. coli M226 (CGSC #4997) lysogenized with λDE3

Growth medium, induction protocol: The construct DNA was transformed into competent cells of the expression strain by a standard heat shock procedure. One colony from the transformation was used to inoculate 1 ml of TB media containing 50µg/ml kanamycin and 34µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day glycerol stocks were prepared from this overnight culture. A glycerol stock was used to inoculate 50ml of TB media containing 50µg/ml kanamycin and 34µg/ml chloramphenicol, which was placed in a 37°C shaker overnight. The next day this starter culture was used to inoculate 3L of TB media (7.5ml starter culture used per 1L) containing 50µg/ml kanamycin. When the OD₆₀₀ reached approximately 0.8 the temperature was reduced to 18°C and after a further 30 minutes the cells were induced by the addition of 0.1 mM IPTG. The expression was continued overnight. Cells were harvested by centrifugation at 6000 x g after which the supernatant was poured out and the cell pellet either placed in a -20°C freezer or used directly for purification.
Lysis buffer: 50 mM HEPES, pH 7.5; 500 mM NaCl; 10 mM Imidazole; 5% Glycerol; 0.5 mM TCEP; 1 tablet per 50 ml protease inhibitor cocktail EDTA-free (Roche).
Extraction buffer, extraction method: Cell pellets were dissolved in approximately 150ml lysis buffer and broken by passing through a high pressure homogenizer at 15,000 psi for 4 cycles. The cell debris was pelleted at 35,000 x g and the supernatant used for further purification.

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

Column 1 Buffers:
Binding buffer : 50 mM HEPES, pH 7.4; 500 mM NaCl; 5% glycerol; 10 mM Imidazole; 0.5 mM TCEP.
Wash buffer : 50 mM HEPES, pH 7.4; 500 mM NaCl; 5% glycerol; 40 mM Imidazole; 0.5 mM TCEP.
Elution buffer : 50 mM HEPES, pH 7.4; 500 mM NaCl; 5% glycerol; 250 mM Imidazole; 0.5 mM TCEP.

Column 1 Procedure: The clarified cell extract was incubated with 1.5ml of Ni-NTA pre-equilibrated with lysis buffer for 1 hour at 4°C with rotation after which it was passed through a glass column. The column was then washed with Binding Buffer (50ml) and Wash Buffer (50ml). The protein was eluted with 25ml of Elution Buffer in 5ml fractions.

Column 2: Superdex 75 16/60 Gel Filtration.

GF Buffer: 10 mM HEPES, pH 7.5; 500 mM NaCl; 5% glycerol, 0.5 mM TCEP.

Column 2 Procedure: The elution buffer fractions from column 1 were pooled and concentrated to 2ml with a 10kDa mwco spin concentrator and injected onto an S75 16/60 column (pre-equilibrated in GF Buffer) at 1.0ml/min. 1ml fractions were collected.

Enzymatic treatment: Protein from fractions eluted at 50-55ml from S75 gel filtration were pooled and incubated with 1:20 mol:mol TEV protease overnight at 4°C. Then protein plus TEV was passed through a column containing 0.5ml Ni-NTA pre-equilibrated with GF Buffer. Column was washed 1ml of GF Buffer. Flow-through and wash fractions were pooled.

Protein concentration: 10.5mg/ml using Millipore 10kDa mwco concentrators.

Mass spectrometry characterization: After TEV protease digestion:
Measured mass: 29583.8Da
Expected mass: 29582.8Da

Crystallisation: Prior to crystallization, protein was pre-incubated with 1 mM MnCl₂ and 1 mM uridine diphosphate UDP. Crystals were grown by vapour diffusion in sitting drop at 20°C. A sitting drop consisting of 100nl protein and 50nl well solution was equilibrated against well solution containing 2 M ammonium sulfate and 0.1 M Bis-Tris pH 6.5. Crystals were mounted in the presence of 25% (v/v) ethylene glycol and flash-cooled in liquid nitrogen.

Data collection:
Resolution: 2.00Å.
X-ray source: FRE superbright, single wavelength.

References

Lomako J, Lomako WM, Whelan WJ (1988) A self-glucosylating protein is the primer for rabbit muscle glycogen biosynthesis. FASEB J 2: 3097-3103
Mu J, Skurat AV, Roach PJ (1997) Glycogenin-2, a novel self-glucosylating protein involved in liver glycogen biosynthesis. J Biol Chem 272: 27589-27597
Moslemi AR, Lindberg C, Nilsson J, Tajsharghi H, Andersson B, Oldfors A (2010) Glycogenin-1 deficiency and inactivated priming of glycogen synthesis. N Engl J Med 362: 1203-1210