Cargèse BIP Marseille EcoleDécembre du GERM 2011 2013
Protein NMR Structure Dynamics Interaction function Bruno Kieffer Biomolecular NMR Team
Modern biology : towards complexity GFP Polymerase Chain Reaction
1995
ESI MS/MS High Througput Sequencing
RNA Seq
2000
Human Genome
2005
2010
Transcriptome Proteome
Interactome Metabolome
Bio-NMR Evolution milestones TROSY (A. Pervushin) K Wüthrich Protein. Solution Str.
1980
15N-13C
labelling Methods (Ad Bax)
Relaxation Dispersion (A. Palmer)
1990
2000
Methyl labelling (L. Kay)
RDC (J Prestegard, N. Tjandra)
Longitudinal Relax. Optimized spectr. SOFAST (P. Schanda)
2010
Protein : a complex polymer • A naïve view of a protein Primary sequence ippdqqrlifagkqledgrtlsdyniqkestlhlvlrlrg gmqifvktltgktitldvepsdtienvkakiqdkegi
3D fold
Protein : a complex polymer • A evolutionnary view
Protein : a complex polymer • A thermodynamic view of a protein Lysosyme pH7.0
From Creighton, proteins Freeman 1993
Protein : a complex polymer • A thermodynamic view of a protein
From Creighton, proteins Freeman 1993
NMR spectrum of a 12 kDa protein H2O
Buffer (TRIS)
aliphatic protons aromatic protons amides protons
Hα Methyls
8
NMR spectrum of a 12 kDa protein H2O
Spectre d une MVKQIESKTA FQEALDAAGD Protéine de 10 kDa
Buffer (TRIS)
KLVVVDFSAT WCGPCKMIKP FFHSLSEKYS NVIFLEVDVD DCQDVASECE VKCMPTFQFF KKGQKVGEFS GANKEKLEAT INELV
Valine 52
aliphatic Valine protons 65
aromatic protons amides protons
Hα Methyls
9
Resonance frequency of nuclei within a protein provide a probe for local environment
Unfolded protein
10
Folded protein
Resonance assignment and labelling HN Hβ, Hγ, Hδ...
2D NOESY spectrum of a 12 kDa protein Spectral assignments are performed thanks to a combination of isotopic labelling schemes and associated NMR experiments
HN Hα
HN HN
Hα Hβ, Hγ, Hδ...
Resonance assignment and labelling
Interaction or Resonance assignment and labelling Dynamics experiments
The 1H-15N HSQC : a relentless judge of the protein’s state
15N
1H
15N
The 1H-15N HSQC : a relentless judge of the protein’s state When defining the structural domain sequence boundaries
The 1H-15N HSQC : a relentless judge of the protein’s state When choosing the affinity purification tag
Measuring 1H-15N HSQC in-cell
Zach Serber, Philipp Selenko, Robert Hänsel, Sina Reckel, Frank Löhr, James E Ferrell, Jr, Gerhard Wagner & Volker Dötsch Nature Protocols 1, 2701 - 2709 (2007)
The use of NMR for protein control quality : the protealys project Aim: Design a method able to analyse therapeutic proteins within complex mixtures
Spectres RMN Spectromètre
Formule
Human insulin from two different companies
umuline Lilly
actrapid NovoNordisk
The proteins are identical The compositions are differents
Detection a single pair of amino-acid inversion Insuline humaine
actrapid NovoNordisk Insuline lispro
humalog NovoNordisk
inversion
15N-13C
labelling : protein backbone roadmap triple resonance experiments
Exp: HNCA
15N-13C
labelling : protein backbone roadmap triple resonance experiments
Identification of residue type The spectroscopic approach F,Y,H,W,C,S G
V,I
A
T
D,N
R,E,Q,K,P,M,L
Hadamac (E. Lescop et al. J. AM. CHEM. SOC. 2008, 130, 5014–5015)
Identification of residue type The spectroscopic approach
Hadamac (E. Lescop et al. J. AM. CHEM. SOC. 2008, 130, 5014–5015)
Identification of residue type The Biochemical approach : selective depletion in cyanobacteria expression system
[
12C
Carbon source: CO2 Energy: light Nitrogen source: NO-3 Inducer : NO-3
- 14N
] Ile
Identification of residue type The Biochemical approach : selective depletion in cyanobacteria expression system Addition of unlabelled ILE
Metabolic leakage
Selective labelling
Constant-time 1H-15N HSQC with N-CO filter
Cell-free expression system prevents metabolic leakage
Cell-free expression and stable isotope labelling strategies for membrane proteins. Sobhanifar S, Reckel S, Junge F, Schwarz D, Kai L, Karbyshev M, Löhr F, Bernhard F, Dötsch V. J Biomol NMR. 2010 p 33-43
J Biomol NMR (2012) 52:197–210
Towards large complexes selective methyl labelling Use of metabolic precursors Leads to both S and R isomers of V and L
Sprangers et al. Solution NMR of supramolecular complexes: providing new insights into function. Nat Meth (2007) vol. 4 (9) pp. 697-703
Hemisynthesis Leads to specifically the pro-S isomer
Stereospecific Isotopic Labeling of Methyl Groups for NMR Spectroscopic Studies of High-Molecular-Weight Proteins** Gans P. ,…, Jérôme Boisbouvier* Angew. Chem. Int. Ed. 2010, 49, 1958 –1962
selective methyl labelling: the malate synthase G (82 kDa)
Assignments of methyl groups using single aminoacid mutagenesis
Amero … Boisbouvier, J Biomol NMR (2011) 50:229–236
Protein structure determination
Improved structure calculation procedures and programs Structure determination from NMR data is an non-linear inverse problem:
Dihedral angles
NOEs
Improved structure calculation procedures and programs Protein dynamics leads to a non-linear averaging of NMR observables
Improved structure calculation procedures and programs Iterative and automated structure determination protocols allow to resolve NOE ambiguous assignments Initial fold
i=1 NOEs assignment, Artefact suppression Medium ambiguity accepted
i=2
... i=8
softwares: ARIA, UNIO
Rieping W., Habeck M., Bardiaux B., Bernard A., Malliavin T.E., Nilges M. (2007) ARIA2: automated NOE assignment and data integration in NMR structure calculation. Bioinformatics 23:381-382. Volk, J.; Herrmann, T.; Wüthrich, K. J. Biomol.NMR. 2008, 41, 127-138..
Use of Ambiguous Interaction Restraints for soft docking
Domingez C, Boelens R, Bonvin A, J. Am. Chem. Soc. 125, 1731-1737 (2003).
Use of Ambiguous Interaction Restraints for soft docking
Probing molecular interactions
KD =
koff kon
koff CH3
kon
+
CH3
Kinetics of the equilibrium defines the echange regime fast
Exchange rate
intermediate
slow
NMR spectrum
νbound
bound lifetime: (k on KD)-1 KD :
fs
ns
νfree
νbound
νfree
νbound
νfree
µs
ms
s
h
mM Weak binders
µM
nM strong binders
Interaction between a small ligand and a protein « by simple NMR » BSA + D-Trp Experimental conditions - Unlabelled BSA 900 uM - 3 mm tube (< 1 nmol prot) - 700 MHz cryo TCI - 2 hours acquisition - SOFAST METHYL-TROSY
08.4
16.6
13C
(ppm)
12.5
20.7
24.8
1.92
1.08
1.50 1H
0.66
0.24
(ppm) Quinternet...Kieffer, Chem. Eur. J. 2012, 18, 3969 – 3974
Fast pulsing rate reduces ligand signal Titration BSA/paracétamol [BSA] = 1.8 mM
I
100
90
14.7
a
23.0
I
[P] = 18 mM
13C
(ppm)
73 ms
P
BSA 100 61
CH3
1200 ms
b P
73 ms
2.18
1H
(ppm)
0.80
2.18
BSA 1H
(ppm)
0.80
(A)
(B) Δδ
Δδ 0.12
(C)
KdL = 35 ± 5 mM
17.1
0.04
type L
0.10 0.01 0.01
0.05
KdH = 45 ± 15 µM
type H 0
4000
13C
0.08
17.4
0
8000 10000 [L-Trp] µM
0
(D)
0
(ppm)
0.15
2000
320 13380 20000
10260
L
High
1280 3200
type C 1H
(ppm)
koff1 High
R
kon2
Low
koff2 High
Low
kon3 koff3
kon4 High
Low
R2L
koff4
960
5130
R1L kon1
640
7060
24450
0.98
10000 20000 [L-Trp] µM
150
Low
RL2
1920
0.91
Low affinity interaction are involved in signalling Example of the Retinoic Acid Receptor
RARγ
RARγ
RAR β
α
RAR RARα
SH3.3 domain of vinexin β
SH3 + RARg NTD peptide titration experiments
SH3-3
+
PSPPSPPPPPRVYK!
Modulation of RARγ activity by A/B phosphorylation P
EEMVPSS77PS79PPPPPRVYK P
S
S
S
S
S
S
S
S
Kd! 77S-79S
: 40 µM 140 µM 77S-79pS : 280 µM 77pS-79pS : 545 µM 77pS-79S :
Lalevee et al. Vinexin , The FASEB Journal (2010) vol. 24 (11) pp. 4523-4534
Chemical shift mapping of RARγ PRD-DBD on vinexin β SH3.3 ∆δ ppm (PRD.DBD – PRD)
SH3 sequence
binding surface
SH3/PRD : KD > 40µM SH3/PRD-DBD : KD = 12µM DBD RARγ
Chemical shift mapping of SH3 interaction site on RARγ DBD 9.5
9.0
8.5
8.0
110
7.5
7.0 110
T68N-HN G39N-HN
Disappearing correlations
K50N-HN G29N-HN
V34N-HN T55N-HN
S47N-HN M52N-HN
N70N-HN
115
S86N-HN
V67N-HN
115
F81N-HN
Q78N-HN S6N-HN
R46N-HN
(ppm)
R45N-HN C23N-HN S9N-HN
C80N-HN
t 1 - 15N
Y32N-HN S28N-HN
C72N-HN
M0N-HN
K60N-HN
R69N-HN
S7N-HN
Y74N-HN E38N-HN
V90N-HN
Y30N-HN
R71N-HN
S27N-HN
E88N-HN
N51N-HN
N24N-HN N61N-HN K66N-HN
Q49N-HN I48N-HN
120
D25N-HN
120
A89N-HN
L77N-HN
Unaffected correlations
E2N-HN
S36N-HN V22N-HN
K41N-HN
M85N-HN
C20N-HN
C37N-HN
E1N-HN R15N-HN
K26N-HN
K79N-HN
V16N-HN
R76N-HN
M3N-HN
F43N-HN
I63N-HN H31N-HN V4N-HN
C75N-HN
E82N-HN
D59N-HN N92N-HN
R91N-HN
Y17N-HN K18N-HN N65N-HN
125
125
C62N-HN
F44N-HN
K87N-HN R58N-HN
9.5
9.0
Shifting correlations
8.5
8.0
7.5
7.0
t 2 - 1H (ppm)
GAMEEMVPSSPSPPPPPRVYKPCFVCNDKSSGYHYG
VSSCEGCKGFFRRSIQKNMVYTCHRDKNCIINKVT RNRCQYCRLQKCFEVGMSKEAVRN
Building the 3D model of the SH3/DBD complex using HADDOCK
What about the DNA ? DNA : RARE DR0 Vinexin SH3.3 PRD-RARγ DBD
Revealing invisible states : Folding upon binding of KIX to KID phosphorylation of KID Ser 133 trigger the formation of a complex between CREB and CBP resulting in enhanced gene expression
Kenji Sugase, H. Jane Dyson & Peter E. Wright (2007) Nature 447, 1021-1025. Mechanism of coupled folding and binding of an intrinsically disordered protein.
Revealing invisible states : Folding upon binding of KIX to KID
Revealing invisible states : Folding upon binding of KIX to KID
fast exchange regime “encoutering” complexes
fast/intermediate three site exchange regime in the bound state
➫ The formation of transient complexes is involved in the protein recognition ➫ A intermediate complex (where αA is folded up to ~90%) is then formed without dissociation.
Evidencing conformer selection processes in allosteric regulation Two model mechanisms for allostery : Induced Fit
Conformational selection
Evidencing conformer selection processes in allosteric regulation
c-Myb MLL KIX !ex = 3.0 ± 0.3ms @ 27°C
➫ Binding of MLL transcription factor to KIX induces a redistribution of population towards conformations where c-Myb (pKID) binding site is preformed. ➫ This minor state is not seen in the free KIX protein Brüschweiler et al, J. Am. Chem. Soc. (2009) 131, 3063–3068
Evidencing conformer selection processes in allosteric regulation
Brüschweiler et al, J. Am. Chem. Soc. (2009) 131, 3063–3068
Evidencing conformer selection processes in allosteric regulation ➫ Identification of a hydrophobic cluster of residues bridging the two binding sites
Brüschweiler et al, J. Am. Chem. Soc. (2009) 131, 3063–3068
Exploring molecular mechanisms within the chromatin using NMR
•
Chromatin has emerged as an active component of gene expression regulation.
•
Post-translational modifications (PTM) of Histones are used to activate/ repress gene expression at specific loci.
Proton spectra of nucleosome preparations Signals from DNA imino-protons
T-A
G-C
•
It is a large particle for NMR (Tc > 100 ns)
•
It contains three distinct spectroscopic "territories" – DNA – Disordered flexible tails – 4 small proteins
Sharp signals from disordered tails
DOSY spectrum of nucleosome preparations from nuclear extracts treated by DNAase
Exploring molecular mechanisms within the chromatin using NMR
From Kato et al PNAS 2011
Mapping the interaction between HMGN2 and the nucleosome
conclusions • NMR allows the investigation of energy landscapes of proteins, which is closely related to their function and their evolution. • Recent methodological developments opened new time windows to explore the dynamics of proteins and their interactions. • There is a need to develop sensitive probes to explore molecular mechanisms in more complex environments.
Plan • Introduction : perspective historique • La HSQC une porte sur l’intimité des protéines • Assignments – Attribution des signaux – Marquage isotopique – Méthodes modernes (cell-free expression systems) • Détermination de structures des protéines – ARIA / Soft docking
• • • •
Etude du repliement In-cell NMR Protéines intrinsèquement dépliée (repliement et interaction) Etudes d’interactions ligand-protéines – Echelles de temps et d’affinité – Observation des ligands – Observation du récepteur: site spécifique – Processus d’auto-assemblage
