NMR for studying biomolecular recognition and dynamics

Transcription

1 NMR for studying biomolecular recognition and dynamics Michael Sattler Outline Biomolecular NMR Tools for studying protein ligand interactions and macromolecular complexes Example Multi-domain proteins and complexes in splicing regulation

2 Structure/imaging from molecules to animals MRI Animal Static picture, snapshots Light microscopy EM tomography Cryo EM SAXS, SANS X-ray NMR Size, spatial resolution Cell Protein complexes Molecular machines 10 3 [s] Proteins, domains Dynamics, timescales Chemical Biology Small molecules Dynamics: regulation NMR Light microscopy MRI Biomolecular NMR Structure determination of biomacromolecules no crystal needed, native-like conditions: solution, macromolecular crowding, in cell NMR (Xenopus oocyctes) transient regulatory interactions, flexible linkers Ligand binding and molecular interactions in solution NMR fingerprint: macromolecular and small molecule interactions Dynamics and mobility (ps days) conformational dynamics enzyme turnover, kinetics, folding Multidisciplinary approaches combine NMR and X-ray with SAXS/SANS, EPR, FRET, free 250 bound 200 T2 [ms] SANS SAXS RDCs residue

3 Nuclear spins, Magnetic moments & Resonance precession frequency B 0 B 0 NMR Magnet Magnetic field nuclear magnetic dipole Nuclear spin Resonance = B 0 Apply radio frequency at resonance to measure the nuclear precession frequencies Fourier transformation frequency NMR spectrum time t 900 MHz Historischer NMR-Magnet (Eisen, 1.4 Tesla, 60 MHz) 60 MHz 250 MHz

4 800 MHz 900 MHz 750 MHz 600 MHz 250MHz Ribonuklease 40 MHz M. Saunders et al. J.Amer.Chem.Soc. 1957, 79, 3289 Lysozym 900 MHz München ppm

5 A 1D NMR spectrum of a protein H H H Is my protein folded? Unfolded 20 kda protein Folded 20 kda protein CH3 aliphatic CH3 CH2 backbone HN backbone HN side chain NH2 aromatic CH Trp H side chain NH2 aromatic CH CH ppm ppm ppm ppm

6 NMR sample Sample preparation and isotope labeling ( 15 N, 13 C, 2 H) overexpression of recombinant proteins in E.coli using minimal media Sample conditions: NMR fingerprint spectra H 2D 1 H, 15 N correlation (HSQC, TROSY) C O N C 15 N 15 N 1 H 1 H Side view Contour plot

7 Chemical exchange A B NMR time scale (chemical shift) slow exchange k ex << coalescence k ex ~ k 1 A B k -1 k ex = k 1 k -1 fast exchange k ex >> Chemical or conformational exchange: Line widths and Larmor frequencies depend on the exchange rates and the chemical shifts of the interconverting states behaves similar to NOE cross peaks in 2D NOESY rate constants can be determined, e.g. 2-state binding equilibrium, chemical reaction, or conformational exchange Dynamics - NMR time scales A B NMR time scale (chemical shift) slow exchange k ex << coalescence k ex ~ k 1 A B k -1 k ex = k 1 k -1 fast exchange k ex >> Chemical or conformational exchange can be analyzed by NMR Rate constants can be determined, e.g. for a 2-state binding equilibrium, chemical reaction, or conformational exchange

8 NMR time scales and dynamics in biology T1, T2 relaxation NOE T1ρ, CPMG exchange spectroscopy Residual dipolar and scalar couplings (RDCs, J) real time rates s s s s -1 1 s -1 time 1 ps 1 ns 1 s 1 ms 1 s molecular tumbling folding internal motion enzyme kinetics, exchange ligand binding bond vibrations side chain rotations domain motion ligand binding enzyme kinetics folding, H/D exchange Ligand binding in NMR titrations - fast exchange Binding in fast exchange on the NMR chemical shift time scale Dissociation constant K D from binding isotherm free bound [P] < K D Fraction bound [PL] ~ obs - free = f([l tot ]) Selenko et al (2001) Nature Struct. Biol. 8, k on P L PL k off K d = [P][L] / [PL] = k B /k A k A = k on [L]; k B = k off B = protein-ligand complex PL A = free protein P

9 The NMR band shift and binding site mapping Chemical shift perturbation upon ligand binding Mapping of the ligand binding site onto the structure Protein Protein-RNA Lingel et al (2003) Nature 426, Lingel et al (2004) Nat Struct Mol Biol 11, Ligand binding by NMR - slow exchange 1:1 B2 dimer:dsrna stoichiometry binding affinity: K d ~ nm (slow exchange) non-sequence specific dsrna contacts (one set of NMR signals, non-palindromic ligand) Binding in slow exchange on the NMR chemical shift time scale [P] > K D 5 GCAGCACGACUUCUUCAAGTT 3 3 TTCGUCGUGCUGAAGAAGUUC 5 Lingel et al. (2005) EMBO Rep. 6,

10 Folding upon ligand binding seen by NMR NMR spectrum of a novel RNA binding domain when bound to an RNA oligonucleotide Protein ProteinRNA (misfolded) Mourao et al. RNA. (2010) Structure-based drug design Targeting protein-protein interactions by small molecules: Structure of protein-peptide complex available Starting point for structure based ligand design In silico screen => NMR hit validation OH N Cl Cl OH Crystal structure of protein-peptide complex In silico screen NMR titrations with small molecule inhibitors

11 SAR by NMR Nature (2005) Peptide binding Bcl-2 inhibitor binding Science (1997) Structural modules at the 3 splice site Selenko et al Mol. Cell. (2003) Kielkopf et al. Cell (2001) Liu, Luyten et al. Science (2001) SF1 P RRM3 U2AF 65 U2AF 35 QUA2 KH RRM 5 UACUAAC UUUUUUU AG 3 Ito et al. EMBO J. (1999); Sickmier et al Mol.Cell (2006)

12 U2AF65 - form a compact structure U2AF65 - necessary and sufficient for Py tract RNA binding Two structural domains, connected by a flexible linker SF1 U2AF 65 U2AF 35 U2AF 65 U2AF 65 UACUAAC UUUUUUU AG Transverse relaxation T2 [ms] Flexibility U2AF 65 - U 9 RNA UUUUUUU flexible linker MHz, T=295K c 12 ns MW 20 kda Residue Number NMR approaches for studying large complexes 3D structure of subunits available (X-ray, NMR, homology model) Subunit-selective labeling, optimized 2 H-labeling Domain interfaces PRE (spin label) Chemical shift perturbations upon binding Interdomain NOEs Saturation transfer PREs (spin labeling) Solvent PREs Solvent PRE Domain arrangement, orientatio Residual dipolar couplings (RDCs) Pseudo contact shifts (PCSs) Small angle scattering (SAS) RDCs Domain orientation Structure calculation Joint refinement against all restraints: CSP, (NOE), PRE, RDC,SAS SAXS/SANS Simon, et al Angew. Chem (2010) ; Madl et al JACS (2010); Madl et al Angew. Chem (2011); Madl et al J Struct Biol (2011)

13 Interdomain distance restraints from spin labels PRE (paramagnetic relaxation enhancement) Long-range distance restraints (<20 Å) Multiple single-cys mutants molecular biology Distance calibration: transverse PRE Reference Spin label RNA spin labeling: Chemically synthesize thiouracil RNA oligo Protein spin labeling: CH 3 CH 3 N O CH 3 CH 3 O NH C CH 2 S N R N 4-thiouracil proxyl Varani JACS (1998) O Recombinant single Cys mutant proteins MTSL IPSL 3-[2-iodoacetamido]-proxyl Global structure from NMR data Individual domain structures available Spin labeling paramagnetic relaxation enhancements (PRE) PRE-derived distance restraints to define interdomain arrangement Interdomain distance restraints IPSL Simon, et al (2010) Angew. Chem; Madl, et al (2010) JACS

14 Solution conformation differs from crystal structure NMR Importance of using solution methods for studying multidomain proteins RDC exp [Hz] X-ray RNA RDC calc [Hz] 2 linker RDC exp [Hz] Sickmier et al Mol. Cell (2006) RDC calc [Hz] Distinct domain arrangement Bound to U9 RNA No RNA residue residue open closed Mackereth et al Nature (2011)

15 U2AF65 - adopts a closed conformation The RNA-binding surface of in free U2AF 65 is partially shielded RNA Chemical shift /ppm K D = 160 M () K D > 3 mm () [U(4)RNA] /μm C305 () G154 () U(4) U2AF 65 U(9) U(13) Model Py-tracts : vs binding Py tract 3 ss Human U2 introns K D /μm 33 UUUU U4 Py tract strength UUUUAAAA U4A4 UUUUAAAAAAAAUUUU U4A8U4 UUUUAAAAUUUU U4A4U4 1.3 UUUUUUUUU U UUUUUUUUUUUUU U13 always binds variable binding

16 Population shift between distinct domain arrangements Kd /μm unbound UUUU U4 UUUUAAAA U4A4 UUUUAAAAAAAAUUUU U4A8U4 UUUUAAAAUUUU U4A4U4 UUUUUUUUU U9 Py tract strength spliceosome assembly In vitro splicing assays in nuclear extract Py tract strength RNA 3 ss MINX ATP NE titration A complex K d - 4 U 4 A 8 U μm 16 μm 7.1 μm 1.3 μm U 4 A 4 U 4 A 4 U WT Sophie Bonnal, Juan Valcarcel

17 Population shift closed/open regulates splicing Population open (bound) conformation Py tract strength Pre-existing bound conformations in free - PRE calculated free RNA bound PRE measured Consistent with free structure Consistent with population of bound form

18 Locked closed mutant (D215R/G319R) shifts the equilibrium Mutations remote from RNA binding interface impair RNA binding and complex A formation destabilize 'open' conformation stabilize 'closed' conformation G319R G319R wt mutant D215R G319R D215R G319R D215R D215R Multi-domain conformational selection

19 Why two distinct domain arrangements? Exposure of conserved surface: additional regulation by protein binding U2AF35, DEK, A1,? - -/ RNA conservation Conclusions NMR is a powerful technique for studying the structure, molecular interactions and dynamics of protein complexes in solution transient interactions, flexible linkers, Label-free, native-like solution conditions in buffer or even in cell or in cell extracts residue-level structural resolution of binding and dynamics! Large complexes can be studied in solution by using optimized methods ( 2 H labeling, TROSY) and integrated structural biology approaches (NMR SAXS/SANS) Multi-domain conformational selection for Py tract RNA recognition by U2AF65

20 Acknowledgements Cameron Mackereth Tobias Madl Bernd Simon Katia Zanier Alexander Gasch Frank Gabel Kostas Tripsianes Helge Meyer Peijian Zou Andre Mourao Anders Friberg Fatiha Kateb Malgosia Duszczyk Lorenzo Corsini Collaborations Sophie Bonnal, Juan Valcarcel (Barcelona) Michael Nilges (Paris) Dmitri Svergun (EMBL Hamburg) Vladimir Rybin (EMBL Heidelberg) Dirk Görlich, Thomas Güttler (Göttingen) Gabi Schramm (Borstel) Jochen Müller-Dieckmann (EMBL Hamburg)