Structural studies of human GBE1 and relevance to APBD

Wyatt YueStructural Genomics Consortium (SGC)

University of Oxford

APBDRF Meeting Dec 2013

Combining structural biology, protein biochemistry to understand inborn errors of metabolism

www.thesgc.org/wyatt wyatt.yue@sgc.ox.ac.uk

• Public-private partnership

• High-throughput structural & chemical biology of human proteins

• Diverse protein families/biology areas

• Open access research model

Studying enzyme shapes

• Enzymes are proteins

- building blocks (amino acids)

- have different shapes

• Shapes can change too!

• Understanding enzyme functions/malfunctions –need to know its shapes

… seeing molecules in action

‘Taking pictures’ of protein structures

1. make proteins of interest (expression, purification)

2. arrange them in order (crystallization)

3. take picture! (x-ray diffraction)

4. develop the film (modelling)

Our work so far …

APBDRF Meeting Dec 2013

Glycogen Synthesis

glycogenin

glycogenin

glycogenin

glycogensynthase

glycogensynthase

branchingenzyme

priming

elongation

branching

GSD type XV

GSD type 0

GSD type IV (liver)Adult Polyglucosan Body Disease

human GYG1 structure

human GBE1 structure

Cartoon impression of glycogen granule

1 702700

Multi-construct approach

E coli structure

6363

7070

7979

c004c005

c000c001c002c003

54

c101c102

c104c105c106c107

c103

c108

1116163838

54

c109c110c111c112

2nd roundSoluble in insect cells

L28P36

Y41R47

c011, c012c013, c014c015, c016c017, c018

3rd roundFine-tune

• Full length and a series of truncations• N- and C-termini nibbling• N-terminal His6 tagged fusion

1st roundInsoluble in E. coli

GBE1 Purification & Crystallization

150

100

75

50

37

2520

15

10

FT BB1 WB1 E1E2

E3BB2 WB2

Pooled

250

GBE1A c011 E1 s200 GF 300812:Sample1Title_UV GBE1A c011 E1 s200 GF 300812:Sample1Title_Fractions GBE1A c011 E1 s200 GF 300812:Sample1Title_Inject GBE1A c011 E1 s200 GF 300812:Sample1Title_Logbook

0

50

100

150

200

250

300

350

mAU

0 20 40 60 80 100 120 140 ml

Sa

mp

le 1

Na

me

Wa

sh

Fra

ctio

n C

olle

cto

r O

utle

t

Fin

ish

Wa

sh

ing

Fra

ctio

n C

olle

cto

r O

utle

t

Sta

rt C

olle

ctin

g F

ractio

ns

Sto

p C

olle

ctin

g F

ractio

ns

A2 A4 A6 A7 A9 A11 B12 B10 B8 B6 B4 B2 C1 C3 C5 C7 C9 C11 D12 D10 D8 D6 D4 D2 E1 E3 E5 E7 E9 E11 F12 F10 F8

Pooled

u/ccut

FTGF BB2

WBEB

BB1

Pooled

Growth from 4L of Baculovirus-infected insect Sf9 cells

1. Ni affinity 2. Size exclusion

3. His-tag removal by TEV4. Second Ni affinity (to

remove uncut protein, TEV)

Concentrated, purified

15% PEG 3350 0.15M sodium succinate

GBE1A-c014 (recombinant Pro37-Leu700 with a TEV-cleavable His6-tag)

Structure determination

• Three structure data:– apo GBE1, GBE1-acarbose and GBE1-Glc7

Overall structureMapping of gbe1 mutationsComplex with sugar chains

Y329 site

1 702

GBE1 mutants

c10638 700

DISEASE

CATALYTICnucelophileacid/base catalyst

Majority are expressed but only p.D357A is soluble.Attempts were made to optimise the purification of p.Y329S.

1 L insect cells, standard purification procedure

Y329S R515H R524Q D357A E412A

Y329SR515HR524QD357AE412A

GBE1A-c201 e129 p014 p015 (6L, Baculo)

250150100

75

50

37

252015

uncut cut

Test TEV Cleavage Concentrated GF Samples

250150100

75

50

37

252015

250150100

75

50

37

252015

Total Soluble FT WB1 WB2 WB3 WB4 WB5 E1 E2GF, concentrated

Ni affinity

p.Y329S mutant protein purification

GBE1A p.Y329S band confirmed by tryptic digestion.

Scale up from 6L insect cell culture

Many metabolic disorders are misfolding defects

Strategy: Develop non-competitive, allosteric PCs?

Pharmacological Chaperones

Yet, their action is counter-intuitive:

They compete with native substrate/cofactor

Disease Protein Chaperone

Fabry GLA DGJ

Gaucher GBA IFG, DNJ

GM1 GLB1 NOEV

GM2 HEXA Pyrimethamine

Pompe GAA DNJ

MPSIII NAGLU 2AcDNJ, 6AcCAS

Fan et al. 1999; Yu et al. 2007; Matsuda et al. 2003; Maegawa et al. 2007; Parenti et al. 2007;Ficko-Blean et al. 2008

Pharmacological Chaperone (PCs)Rescue of stability and function

Examples of PC development

Next Action

• Scale up less soluble proteins

– WT full-length protein

– Mutants Y329S, R515H, R524Q

• Characterize hits/peptides (Tropak/Kakhlon)

– DSF, limited proteolysis

– Aggregation/unfolding assay

– co-crystallization, ITC

Different tags/fusion proteins to improve solubility

Time, protein consuming

Future: collaboration with APBD

APBDRF Meeting Dec 2013

Working with rare disease patient groups

Collaborative projectsFunded personnel

Inborn errors of metabolism Rare cancers & developmental disordersMembrane proteins & rare diseasesGenomic variation & disease

‘Stone man syndrome’

Public engagementInformation

Alex Bullock

What can we do to help APBDRF?

xtal soaking

Crystals, 3D StructureWT/mutant protein

in silico Docking

Binding, BiochemistryDSF, ITC, BLI, co-xtal

dose response, affinity

hit finding

validation

Mode of Actionfolding, proteolysis, aggregation

Effects on activity, stability

characterization

HT Compound Screening (Michael Tropak)

in silico Ligand Design (peptides – Or Kakhlon)

Rescue in vivo?

patient fibroblast cellsAnimal model (e.g. mouse)

enzyme activity

Cellular Assays

Test Mutantsrescue stability?

Effects on activity

Clinical ‘know-how’

Goal: To develop compounds into a pharmacological chaperone treatment

Example of our In Vitro capabilities

Allosteric domain(AdoMet)

Catalytic domain(PLP, haem)

Pathfinder Awards for Orphan Diseases

human CBS structure

Developed In vitro assays to deconvolute binding modes

Recombinant proteinStructural Biology

Compound libraryCellular assay

Structurally diverse, drug-like hits

kp = 0.381 ± 0.037 min-1

Limited proteolysisIn silico docking Domain mappingFragment DSF

Aim: look for binders at different pockets/regions as chemical starting point

Cystathionine beta synthase (CBS) deficiency

ACKNOWLEDGEMENTSTHE MOBThomas McCorvieDipali PatelJolanta KopecStephanie OerumFiona FitzpatrickSean FroeseWasim Kiyani

CRYSTALLOGRAPHY

Frank von DelftTobias Krojer

BIOTECHNOLOGY

Claire DamerellPravin Mahajan

FUNDING PARTNERS

The Canadian Institutes for Health Research, the Canada Foundation for Innovation, Genome Canada, GlaxoSmithKline, Lilly Canada, the Novartis Research Foundation, Pfizer, Takeda, the Ontario Ministry of Economic Development and Innovation, and the Wellcome Trust.

wyatt.yue@sgc.ox.ac.ukwww.thesgc.org/wyatt

2013

2012

Structure determination

• Three structure data:– apo GBE1, GBE1-acarbose and GBE1-Glc7

Overall structureMapping of gbe1 mutationsComplex with sugar chains

Y329 site

Updates on DSF

APBDRF Meeting Dec 2013

Differential Scanning Fluorimetry (DSF), ‘Tm shift’ Niesen et al 2007 Nat Methods

WT

Thermal stability as a ligand binding assay

Destabilization due to mutation – left shift (- Tm)

Stabilization by native ligands –right shift (+ Tm)

Small molecule stabilization? –right shift towards WT

DSF considerations for GBE1

Tm = 41.8 ± 2.1 °CN=8 experiments

N=2 experimentsNo detectable Tm shift with various sugar analogues

- Even at higher concentrations as tested previously- despite electron density in structures! - These are not natural ligand but shortened versions

Tm for apo protein varies with experiments - error margins large, with experiments/preps- is stability concentration dependent?

Is conformational change required for catalysis?(This increases likelihood of seeing Tm shift) - Compounds may not be ‘large’ enough a?

Difference between Full-length vs truncated proteins(previous) E. coli expressed, FL protein - Tm varies with constructs- ->N-terminal may be important for stability/disease? - may explain lack of detectable binding?

Acarbose (4)Glc4,Glc7

4-mer peptides

1702

c105c106c011

700

c013c014

3838

283636

41c015

p001 = cut c105 usedp002 = cut c106 usedp003 = cut c106 usedp004 = uncut c106 ~200ul 16.5mg/mlp005 = cut c106 ~100ul 2.2mg/mlp006 = cut c011 ~120ul 6.9mg/ml*p007 = cut c014 ~530ul 16.1mg/ml

*p009 = uncut c013 ~550ul 20.2mg/mlp010 = cut c013 ~330ul 3.5mg/mlp011 = uncut c015 ~250ul 7.3mg/mlp012 = cut c015 ~80ul 7.3mg/mlp013 = uncut c106 ~50ul 10.7mg/ml

702700

702702

700702

02 Oct 2012, 15 Jan 2013

15 Sep 2013

GBE1 constructs purified

Y329 forms hydrogen bond between its side-chain and backbone carbonyl of His289.2.5 Å distance in WT increases to 8.5 Å in p.Y329S.

Virus re-amplified , extraction buffer optimised to sodium phosphate based,

Talon (cobalt) used instead of Ni-NTA (Nickel).

WT p.Y329S

Structural analysis of Y329 site

Structure determination

• Three structure data:– apo GBE1, GBE1-acarbose and GBE1-Glc7