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Isabelle Krimm | FBDD 2014 | NMR Applications in FBDD regarding fragment binding mode and fragment-induced protein conformational change
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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
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