Complexes de lanthanides pour la biologie structurale

Porquerolles, 10-12 Octobre 2018

François Riobé

Chemistry for optics team

2

Chem. Eur. J., 2017

Luminescent materials

Functional bioprobes

Link luminescence ↔ magnetism

LnIII

Chelating ligand

Energy transfer Emission

Excitation

Protein crystallography

J. Am. Chem. Soc., 2017

In preparation

3D structure of proteins• Up to 11.000 structures a year

• NMR (9%)• requires an enrichment N15 C13

• Crystallography (90%)• Direct access to the structure• Needs diffracting crystals• Solving the phase problem… 3

1985 1990 1995 2000 2005 2010 20150

20000

40000

60000

80000

100000

120000

140000

Stru

ctur

es

Total Yearly

X-RAYNMRELECTRON MICROSCOPYHYBRIDother

source: RCSB Protein Data Bank, May 2017

ESRF, Grenoble, FRANCE

Challenges in crystallography of proteins• Only 27 % of purified soluble proteins give diffracting crystals

4

0

10000

20000

30000

40000

50000

60000

70000

purified crystallized diffractingcrystals

crystalstructures

Source: PSI Structural Biology Knowledgebase, May 2016

Standardization of crystallization conditions

Robotized crystallization platforms

Crystallization platforms and large instruments• More efficient light sources

• Automation of the collect

• Towards high-throughput crystallization projects• Looking for extreme environment (pressure, µgravity...)

5

European XFEL

Crystallization platforms and large instruments• More efficient light sources

• Automation of the collect

• Towards high-throughput crystallization project• Looking for extreme environment (pressure, µgravity...)

6

European XFEL

Thomas Pesquet handling protein crystallization facility hardware on the ISS for a crystallization investigation by Merck & Co.

Challenges in crystallography of proteins• Only 27 % of purified soluble proteins give diffracting crystals

• 50 % of diffracting crystals allow structure determination

• In the end, only 10% of protein structures are determined

7

0

10000

20000

30000

40000

50000

60000

70000

purified crystallized diffractingcrystals

crystalstructures

Source: PSI Structural Biology Knowledgebase, May 2016

“phase problem”

Solving the phase problem

• Anomalous scattering from heavy elements in the structure

• Selenation of methionine residues• Requires purification and crystallization of the selenated protein• Tedious and time consuming method

• Introduction of a lanthanide• By cocrystallization or soaking of a Ln salt

• Strong anomalous signal• May change or even destroy the 3D structure

• Use of MRI contrast agents

8

J. Reuben, Naturwissenschaften1975, 62, 172 – 178.

coll. E. Girard, R. Kahn

Lanthanide complexes for anomalous scattering

• Luminescent lanthanide complexes

• Co-crystals obtained with HEWL

9

[Tb(DPA)3]3-

Sensitization of terbium (II)through ligand absorption

[Tb] et [Eu]@HEWL under UV light

[Tb(DPA)3]3- HEWL co-crystalAngew. Chem. Int. Ed. 2008, 47, 3388

Supramolecular binding

10Specific supramolecular interactions with arginine residues

NH

*NH

N

N

O

O

H H

H

HProtein

N

O O

OO

LnN

OO

OO

NO

OO

O3

Phys. Chem. Chem. Phys., 2013,15, 18235.

Collab. E. Dumont (ENS Lyon)N. Giraud (ICMMO, Paris Sud)

Instability of [Ln(DPA)3]3- in screening kits

11

C84H140Ca6Eu4N12O100

Alcaline-earths Transition metals

Neutral complex

low solubility in water no electrostatic interaction

with proteins

N

N

OO

N NO

O

N

N

O

O

Tb

neutral

Instability of [Ln(DPA)3]3- in screening kits

12

C84H140Ca6Eu4N12O100

Alcaline-earths Transition metals

N

N

OO

N

N

N

O

O

Tb

H

+

Cl-

Charged: 1+Inner coordination sphere incomplete

High solubility Affinity towards negatively charged areas? Recognition of carboxylate?

Effet Nucleating effect

13

HEWL (129AA) crystallisation diagrams

native conditions

After 2 days

clear drop crystals

Nucleating effect

14

HEWL (129AA) crystallisation diagrams

native conditions with 10 mM of Tb-XO4

After 2 days

clear drop crystals

Nucleating effect

15

HEWL (129AA) crystallisation diagrams

native conditions with 10 mM of Tb-XO4

After 2 days

After 20 days

clear drop crystals

Nucleating effect• Test panel of 8 proteins

16

Nucleating effect• Comparative experiment without (native) and with 10 mM Xo4

• 6 crystallization screens for 576 conditions:• PEGs-I Quiagen• Salt-grid HR,• Wizard I-II Rigaku• PACT MD • CSG MD • Classics Suite Qiagen

• Screening performed at the HTX-Lab platform (EMBL-Grenoble)

• 100 nL of protein solution with 100 nL of precipitant solution

17

Nucleating effect

18

• Number of crystallization hits over 576 conditions after 34 days

Nucleating effect

19• Xo4 provides new unique conditions!

Shared conditions

Native conditions only

Xo4 unique conditions

Nucleating effect

20

MDH pb9

+ Xo4

native native

+ Xo4

• Xo4 provides new unique conditions!

Chem. Sci. 2017, 8, 5909-5917.

Nucleating effect

• Results after 7 days: clear drop crystals precipitate

• Xo4 allows crystal growth at low protein concentration• At 5 mg/mL, crystals are ready to use for X-ray diffraction

21

Pb6: shared crystallization condition HEPES pH 7.5 100 mM / PEG 6000 11 to 16 %

native conditions with 10 mM of Tb-XO4

Chem. Sci. 2017, 8, 5909-5917.

2 complexes per monomerOccupancy after soaking 100 mM

Tb1: 0.8Tb2: 0.3

Direct coordination with Asp 101 (-15 kcal.mol-1)Hydrophobic cage Trp 62 and 123 (-20 kcal.mol-1 each)

Binding sites: HEWL

22

Chem. Eur. J. 2018, 24, 9739-9746.

Binding sites: Protease 1 Protease 1 from P. Horikhoshiicrystallized in sulfate buffer

23

166 AA per monomer3 monomers in the ASU1 complex per monomer

1 complex per monomer The complex is “neutralised” by a SO4

2-

Interaction with Glu 36, Phe 35 and Arg 75No direct coordination with Asp or Glu

Chem. Eur. J. 2018, 24, 9739-9746.

Binding sites: Protease 1 Protease 1 from P. Horikhoshiicrystallized in sulfate buffer

24

166 AA per monomer3 monomers in the ASU1 complex per monomer

The buffer influence the binding efficiency but not the nucleation ability

Chem. Eur. J. 2018, 24, 9739-9746.

Binding sites: FprA

25

1 site with a full occupancy.

Coordination of Asp 113 on Tb (-24 kcal.mol-1) H-bonding of Gua 399 with a picolinate (-19 kcal.mol-1) interaction of Tyr 382 with pyridine (-16 kcal.mol-1

Multiple and complementary interactions

Unique condition in presence of Tb-Xo4

Seeking new nucleating agents

26

N

N

N

NN

N NNN

HTb

+

Cl-

N N

NN N

N

P

N

NP N

HTb

+

Cl-

MeOO

OMe

O

no effectweak effect

moderate nucleation effect

Patent No. PCT/FR2016/053539

Luminescence properties

• MDH crystallization:

• Protein crystal detection• Discrimination from salt crystals• Could be helpful for crystal centering on the

beamline 27UV led and optical fiber

Salt crystal MDH crystals

under UVunder UV

Seeking new nucleating agents

28

Screening of HEWL with 10 mM of Tb-Xo4 (unique conditions)

Seeking new nucleating agents

29

Same screening with 10 mM of Lu-Xo4

How does it promote nucleation?

Phasing sites only partially relevant Low occupancies upon nucleation conditions (low concentration) Only few crystallographic sites are clearly responsible of an overall

stabilization of the packing Informative to understand interactions prot./complex

Interactions in solution?paramagnetic H-NMR and Dosy-NMRDLS/SAXS

Hypothesis Reduction of surface entropy Stabilization of dangling chains Crystallization of mutants

Localized and versatile screening of repulsive interactions30

Funding:

Sebastiano Di PietroSandrine Denis-QuanquinDelphine PitratJean-Christophe MulatierElise DumontOlivier Maury

Sylvain EngilbergeLouise LassalleCécile BreytonCharles ArnaudEric Girard

Nicolas Giraud

Chemistry for Optics team

Ln23 project31