NMR APPLICATIONS IN FBDD REGARDING  FRAGMENT BINDING MODE AND FRAGMENT-INDUCED PROTEIN CONFORMATIONAL CHANGE

Isabelle KRIMM, University of Lyon, FRANCE

Binding mode of fragments by NMR

¨  NOEs, sparse NOEs ¨  Paramagnetic-induced pseudo contact shifts ¨  STD (epitope mapping) and WaterLOGSY ? ¨  INPHARMA ? (better for binding site) ¨  Chemical shift perturbations observed on protein

spectra

Chemical Shifts in proteins

§  Secondary structures §  Torsion angles §  Aromatic rings §  Oxidation states §  Flexibility §  Solvant, temperature, pH

Isotropic (anisotropic) diamagnetic (paramagnetic) chemical shifts:

Calculation of protein structures using chemical shifts only

Calculation of chemical shifts from protein structures

§  SHIFTS, SHIFTX, SPARTA…

§  http://www.cs23d.ca/ §  http://spin.niddk.nih.gov/bax/software/CSROSETTA/

Calculation of ligand-induced CSP

Chemical Shifts in proteins

Backbone and side chain torsional contribution

Ring current contribution

Hydrogen bond contribution

Electric field contribution

Other contributions

Local charge effect

O- N+

Solvent Temperature

δ = δrc + δtor + δside + δring +δanis + δHB + δe + δother

Anisotropy contribution

C C

+

+

-- C O

+

+

-- C C +

-

-

H+ HHH

!H=5-7 ppm !H=9-10 ppm !H=2-3 ppm

Ligand-induced Chemical Shifts Perturbations

δ = δrc + δtor + δside + δring +δanis + δHB + δe + δother

Binding contributions All contributions except the hydrogen bonds can be simulated

Ligand binding, no conformational change:

Ligand-induced Chemical Shifts Perturbations

2000

McCoy Wyss

2002

McCoy Wyss

2004

McCoy Wyss

2008

Hunter Packer et al.

2008

Stark Powers

2009

Hunter Packer et al.

2009

Gonzalez-Ruiz

Gohlke

•  2000: First use of quantitative CSP for protein-ligand complexes Docking è CSPsim for each docked poseè comparison CSPsim vs CSPexp Calculation of ring current effects using SHIFTS (ligand = amino acid) •  2002: Jsurf (ligand ring center identification)

•  2004 : Jsurf + comparison CSPsim vs CSPexp

2000

McCoy Wyss

2002

McCoy Wyss

2004

McCoy Wyss

2008

Hunter Packer et al.

2008

Stark Powers

2009

Hunter Packer et al.

2009

Gonzalez-Ruiz

Gohlke

•  2008, 2009: SHIFTY calculation of anisotropy, electric field and ring current contributions Jsurf è Docking è CSPsim using shifty è CSPexp comparison è Binding mode

Ligand-induced Chemical Shifts Perturbations

1999

Hunter Packer

2000

McCoy Wyss

2002

McCoy Wyss

2004

McCoy Wyss

2008

Hunter Packer et al.

2008

Stark Powers

2009

Hunter Packer et al.

2009

Gonzalez-Ruiz

Gohlke

•  2009: Guided docking (CSP data included for the scoring function) calculation of ring current contribution

Ligand-induced Chemical Shifts Perturbations

30 60 90 120

1.5

160

1.0

0

-0.5

CS

PN (

ppm

)0.5

-1.0

B

-1.5

15N-HSQC spectra

C

CS

PH

+N (

pp

m)

residue number30 60 120 160

0.00

0.06

0.16

6 4 1 5

CSP magnitudes and signs 9.2

9.2

9.0

9.0

8.8

8.8

8.6

8.6

t2 -

1H (ppm)

116 116

115 115

114 114

113 113

t1 -

15N

(p

pm

)

9.2 9.0 8.8 8.6

113

114

115

116

1H (ppm)

15

N (

pp

m)

CSPH

CSP

N

Free bound

CSP magnitudes

CSP

C

SP

Residue number

Ligand-induced Chemical Shifts Perturbations

1H (ppm)

Ring current effect (ligand-induced)

Experimental CSP Calculated CSP

9.2

9.2

9.0

9.0

8.8

8.8

8.6

8.6

t2 -

1H (ppm)

116 116

115 115

114 114

113 113

t1 -

15N

(p

pm

)

9.2 9.0 8.8 8.6

113

114

115

116

1H (ppm)

15

N (

pp

m)

30 60 90 120

0.2

160

0.1

0

-0.1

CS

PH (

pp

m)

1.5

A

-0.2

Ligand-induced Chemical Shifts Perturbations

CSPHN- filter (AutoDock)

3 binding modes

3 binding modes

2 binding modes

Orientation of the ring BUT

(CSPHN+ STD) – filter (AutoDock)

X-Ray X-Ray NMR

Co-Crystallisation Soaking

Soaking Soaking

(CSPHN+ STD) – filter (AutoDock)

CSP- Guided docking (PLANTS)

I119-HD

I119-HG

A78-HB

13C-HSQC

15N-HSQC

HN Haliph

CSP- Guided docking (PLANTS)

More problematic More problematic

More problematic

CSP- Guided docking (PLANTS)

More problematic

CSP- Guided docking (PLANTS)

What about conformational changes?

KD 300 µM

Bcl-xL

Experimental CSP

Comparison with calculated CSP

The case of Bcl-xL

Experimental CSP Calculated CSP

The case of Bcl-xL

The case of Bcl-xL

Experimental CSP Calculated CSP for 200 docked positions CSP disagreements

22

Free protein

Residual Dipolar couplings (bound protein)

The case of Bcl-xL

RDC disagreements

Best agreement Inhibitor-bound

protein

Free Protein

Bound Protein

The case of Bcl-xL

Experimental structure

CSP Ha-guided Docking CSP HN-guided Docking

CSP Ha-filtered Docking

Docked structure (PLANTS)

CSP HN-filtered Docking

The case of the PCAF Bromodomain

Experimental CSP Calculated CSP for 1000 docked positions

The case of the PCAF Bromodomain

The case of the PCAF Bromodomain

Experimental structure

CSP HN-guided Docking – Y809 CSP HN-guided Docking – 808-814 CSP HN-filtered Docking – Y809

Quantitative analysis of protein CSP for binding mode ?

Comparison of experimental CSP with back-calculated CSP with AutoDock, with PLANTS

§  Conformational change

§  Binding mode Might require other NMR data (STD) if HN are used Ha are the best probes

Conclusion

Thank you

Clémentine Aguirre Tim tenBrink Olivier Cala Béatrice Brion Dany Davesne

Thomas E. Exner Jean-François Guichou