Protein-protein interactions by NMR

Transcription

1 Protein-protein interactions by NMR

2 Fast k on,off >> (ν free - ν bound ) A + B k on k off AB k on,off ~ (ν free - ν bound ) Slow k on,off << (ν free - ν bound ) ν free ν bound

3 Measure K d by e.g. fluorescence, ITC, Biacore, NMR Minimize interacting region, especially of peptides e.g. limited proteolysis Find conditions where both components are stable and interaction still occurs (e.g. salt dependence?) and exchange regime is favourable (change temperature?) Add one component in excess to saturate the other component (excess component usually the unlabelled one)

4 Structures of protein complexes Dynamics of interactions Mapping interactions e.g. for mutagenesis studies Extracting distance information Docking strong strong weak or strong weak or strong weak or strong

5 How do we go about studying weak interactions? Chemical shift mapping may be possible even in intermediate exchange regime If structures of components are known can model the complex Distance information can be extracted using transferred NOE

6 Transferred NOEs Determine the structure of a small ligand binding large molecule Ligand is in excess over protein (improved sensitivity) Intra-ligand NOEs are detected in the bound state by transferring them by chemical exchange to the free state, where they can be observed Requirement is that off-rate is fast: the ligand must associate/dissociate a few times during the mixing time Transferred NOEs are larger than free intra-ligand NOEs and have the same sign as diagonal peaks Intermolecular NOEs are low intensity due to low protein concentration Clore & Gronenborn: J. Mag. Res ; J. Mag. Res Ni & Scheraga Acc. Chem. Res (review)

7 Studying strong interactions: structures of complexes 2 types of experiment: Edited or separated: keep 1 H attached to nucleus X Filtered or rejected: keep 1 H NOT attached to nucleus X

8 Differential Labelling 13 C 13 C, 15 N-protein Unlabelled peptide H 13 C H 14 N H H 15 N 12 C H H 12 C 13 C-edited NOESY NOEs between 13 C- 1 H and all other 1 H ( 13 C- 1 H, 12 C- 1 H, 15 N- 1 H, 14 N- 1 H) X-filtered, 13 C-edited NOESY NOEs between 13 C- 1 H and 12 C- 1 H/ 15 N- 1 H Double-filtered NOESY NOEs between 12 C- 1 H and 12 C- 1 H

9 Considerations when working with complexes Multiple samples required with differential labelling patterns - cost and time implications Sensitivity of experiments e.g. double-filtered experiments, X- filtered, X-edited experiments Unlabelled peptide + large protein, peptide assignment may be problematic Record twice as many spectra Spectral simplification via labelling patterns Inter- vs intra-molecular NOEs

10 Tight Complexes: Slow Exchange Chemical Shift Assignments 1. Backbone experiments: HSQC, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NH recorded on each component of complex 2. Sidechain experiments: 15 N-separated TOCSY, HCCH-TOCSY CC(CO)NH, H(CCCO)NH, HBHA(CBCA)(CO)NH on each component of the complex 3. Coupling constant experiments: HNHA on each component of the complex

11 Tight Complexes: Slow Exchange NOE Assignments 4. Intra- and inter-molecular NOEs mixed: 3D 13 C-separated NOESY 3D 15 N-separated NOESY 5. Intermolecular NOEs: X-filtered experiments. 3D 13 C-separated if sensitivity is good or 2D 1 H/ 1 H version (Zhwalen et al JACS )

12 X-filtered Experiments Two basic types: a) Schemes where X-attached 1 H are removed directly 1 H X 1/2J e.g. generate MQC b) Schemes where interleaved experiments are recorded and have to be added or subtracted to keep or discard X-attached 1 H 1 H X X-pulse present or absent in alternating experiments

13 X-filter (I) Purge sequence X G G1 G1 y G2 1 H 1 2 I = 1 H attached to X nucleus H = all other 1 H 90 ( 1 H), 1 Hz + Iz -Hy - Iy cosπj IS 1 + 2IxSzsinπJ IS 1 ( 1 = 1/2J IS ) -Hy + 2IxSz 90 (X) -Hy - 2IxSy 1. 1 and 2 can be tuned for different values of J IS e.g Hz (aliphatic) and Hz (aromatic) 2. Can add spin-lock (y) on 1 H to remove Ix-based antiphase that escapes X pulses 3. Replaces a 1 H 90 pulse in a sequence

14 X-filter (II) Refocussed half filter 1 H 1 1 X G G1 G1 I = 1 H attached to X nucleus H = all other 1 H 90 ( 1 H) Hz + Iz -Hy - Iy 1 -Hy -IycosπJ IS 1 + 2IxSzsinπJ IS 1 ( 1 = 1/2J IS ) 180 ( 1 H,X) Hy - 2IxSz 1 Hy - Iy A 180 ( OR 1 H) Hy + 2IxSz 1 Hy + Iy B A + B = Hy only (filtered) A - B = Iy only (edited)

15 All 13 C-filtered experiments suffer because 1 J CH varies: Hz aliphatic CH; Hz aromatic CH 1 delays are inevitably only tuned to one J coupling J CH (Hz) aliphatic His Tyr, Phe, Trp δ 13 C (ppm) 1 J CH = (0.365 ± 0.01 Hz/ppm) δc ± 0.5 Hz Zwahlen et al (1997) JACS

16 Adiabatic pulses Trajectory of 13 C magnetization follows effective field for duration of the pulse Carrier frequency of the pulse starts far upfield, sweeps through resonance, then downfield 13 C resonate at different frequencies so they are all inverted at different times, depending on their frequency and the sweep rate and duration of the inversion pulse

17 1 H X G Hy + Iy a G1 1 /2 From a to b: IzcosπJ IS ( 1-2t) = 0 for all spins 1-2t = 1/(2 1 J CH ) (i) X-filter (III) Purge scheme with adiabatic pulse t 1 /2-t G1 b G2 Hy + IycosπJ IS ( 1-2t) - 2IxSzsinπJ IS ( 1-2t) Hz + IzcosπJ IS ( 1-2t) - 2IxSzsinπJ IS ( 1-2t) 13 C spin inverted at time t (t 0) after 1 H 180 pulse 1 J CH evolution occurs for a time 1-2t 1 J CH and t vary for each 13 C nucleus removed by G2 Since t 0 and 1 J CH is smallest for upfield 13 C (i.e. methyls), these must be inverted first. 1 is set close to 1/2 1 J CH (methyl) 1 J CH = Aδ C + B (ii) (A=0.365 Hz/ppm; B = Hz) Frequency of the transmitter δ RF (t) = δc. Substitute for 1 J CH in (i) 1-2t = 1/[2(Aδ RF + B)] time derivative to get sweep rate during WURST

18 3D 13 C-filtered, 13 C-edited NOESY-HSQC Two of these modules at the start of the sequence (shorter than the original half-filter) Suppression factors are fold Putting filter modules at the start of the sequence makes H 2 O suppression easier Filters add to the length of the sequence, so semi-ct, concatenating 1 H chemical shift evolution with the half filter 15 N filter at the same time as the 13 C filter

19 Cdc42-21 kda small G protein of the Rho family GTP cofactor replaced by GMPPNP PAK - 5kDa fragment - can be expressed as GST-fusion K d ~ 30nM Samples: 15 N Cdc42 + unlabelled PAK 15 N, 13 C Cdc42 + unlabelled PAK 15 N PAK + unlabelled Cdc42 15 N, 13 C PAK + unlabelled Cdc42 no deuteration required - all experiments worked 3D X-filtered, 13 C-edited NOESY ran on 13 C, 15 N PAK, unlabelled Cdc42

20 Summary of Cdc42/PAK Intermolecular NOEs Unambiguous Ambiguous 13 C-filter/ 13 C-edited C-NOESY (PAK) C-NOESY (Cdc42) N-NOESY (PAK) N-NOESY (Cdc42) 7 12 Total Intermolecular Total number of NOEs = 4,000

21 Assignment of Unlabelled Component in Complex Filtered/rejected experiments: to assign unlabelled peptide with labelled protein - sensitivity/water suppression poor double filter (ω1 and ω2) required in NOESY but not in through-bond experiments J(CH,NH)-separated NOESY - - better sensitivity and water behaviour (Nietlispach)

22 Double half-filtered NOESY (Otting & Wüthrich 1990 Q. Rev. Biophys ) 1 H 1 1 t1 τ mix 2 2 Acq X ψ 1 ψ 2 Dec ψ = x: Hy - Iy ψ = -x: Hy + Iy A B C D 12 C -> 12 C 12 C -> 13 C 13 C -> 12 C 13 C -> 13 C ψ 1 ψ 2 x x ψ 1 ψ 2 -x x ψ 1 ψ 2 x -x ψ 1 ψ 2 -x -x Individual datasets can be scaled to offset effects of e.g. low labelling efficiency Combination sub-spectrum ω1/ω2 (A+B) + (C+D) ω1-filter,ω2-filter unlabelled/unlabelled (A+B) - (C+D) ω1-filter,ω2-edit unlabelled/labelled (A-B) + (C-D) ω1-edit,ω2-filter labelled/unlabelled (A-B) - (C-D) ω1-edit,ω2-edit labelled/labelled

23 J(CH,NH)-separated NOESY (based on Melacini, JACS ) After the semi-ct period, required magnetization is: -Iy cosπjt2 cosω H t1 modulated by J in t2 13 C-bound 1 H are at ±J CH /2; 12 C-bound 1 H are at J=0 J=0 plane contains intra-peptide NOEs and peptide(f1) to protein (f3) NOEs Intra-protein NOEs and protein (f1) to peptide (f3) NOEs are at ±J CH /2 Intermolecular NOEs in J=0 plane are coupled in F3 Intra-peptide NOEs have a return peak in the same plane

24 Chromodomain complex with methylated peptide from histone H3 Problematic assignment of Me 2 -Lys - not J coupled to anything else Assignment of NOEs from Me 2 -Lys to aromatic residues in the protein (distinguish from Me-NH NOEs by coupling constant) Me-Me NOE in J=0 plane (confirmed assignment of Me 2 - Lys)

25 F3: J =0 Hz 160Hz Phe 45 Trp 42 Me-K9 x F3: J =160 Hz Trp 42 Me-K9 Phe 45 1 H [ppm]

26 Structure calculation strategies If starting from two extended chains with ideal geometry make sure they are not lying on top of each other (bias starting structures) Assignment of intra-molecular NOEs in 13 C- and 15 N-edited spectra: check that they do not have any inter-molecular possibility (they may not appear in X-filter, 13 C-edited experiment if they are weak) Ambiguous NOEs that appear in 13 C-edited NOESY as well as X-filter, 13 C-edited NOESY should be treated in the same way as other ambiguous NOEs as they may contain intra-molecular contribution to the intensity Ambiguous NOEs: the possibilities must be edited for the different experiment types e.g. 13 C-edited NOESY: F1/F3 = 13 CH only; F2 = any 1 H

27 SH3/peptide Calculation Strategy All intermolecular distance restraints were ambiguous (32 from 13 C- filtered/ 13 C-edited experiment) NOE tables generated using AZARA connect and then put into ARIA Ambiguous restraints analysed at each iteration to generate distance restraint tables for next iteration with reduced ambiguity

28 NOEs used in SH3/peptide Structure Calculations Unambiguous Ambiguous 13 C-filter C-NOESY (SH3) N-NOESY (SH3) H NOESYs Totals At end of calculation - 66 intermolecular NOEs

29 Using NMR data for docking Structures of components are known Use NMR data to map binding contacts or determine relative orientation of components Dynamics, allowing only interfaces to move

30 How do you decide which residues are in the contact site? Pick all the peaks in free and bound and calculate combined shift difference in 15 N and 1 H shifts, often defined as: [( 15 N) 2 x 10( 1 H) 2 ] 1/2 Define significant chemical shift perturbation (>1 SD from average shift change) - add in the ones that have disappeared completely Check for solvent accessibility (e.g. NACCESS): NHs that are completely buried are unlikely to be involved in the interaction but are experiencing secondary effects, unless there is a significant structural change (should be obvious from the HSQC). NACCESS cutoff: if the residue is more than 50% exposed it is available for interaction.

31 Using Chemical Shift Mapping Data for Docking (HADDOCK) Take significant shift changes, screened by solvent accessibility Active residues Take residues close to active residues on the surface Passive residues Ambiguous interactive restraint (AIR) N atoms N res B N atoms ( ) (-1/6) d iab = Σ Σ Σ m ia =1 k=1 n kb =1 1 d 6 m ia n kb between any atom m of active residue i in protein A (m ia ) and any atom n of both active and passive residues k (N res total) of protein B (n kb ) and inversely for protein B. For each active residue (i) in A, restraint to any active or passive residue (k) in B, over all atoms and vice versa. Dominguez et al (2003) J Am Chem Soc

32 HADDOCK calculation strategy (i) Randomization of orientations and rigid body energy minimization (ii) Semi-rigid simulated annealing (iii) Refinement with explicit solvent Models clustered by interaction energies (E elec,e vdw,e AIR ) and average buried surface area Lowest energy cluster with the highest buried surface area assumed to be correct Can also add RDCs, inter-molecular NOEs and radius of gyration term to prevent expansion at the interface (Clore(2003) JACS )

33 Cross-saturation Takahashi et al (2000) Nat. Struc. Biol Protein II Protein I Strong binding case R.F. -NH -CH - 15 NH -C 2 H Band-selective WURST saturation, followed by TROSY-HSQC cross-saturation Saturate the aliphatics of protein II - magnetization transferred by spin diffusion to the aromatics and amides. If saturation does not leak to H 2 O, it does not affect amides of protein I. Cross-saturation to the 15 NH in the interface on protein I Measure intensity in HSQC vs time of saturation to find residues in interface More precise than chemical shift mapping

34 Symmetric oligomers by NMR NMR spectra are identical for each monomer Cannot distinguish intra- and inter-monomer distance contacts Breaking symmetry - mixed labelling (unfold/refold?) higher order oligomers may have to use tags to get single subunit labelled addition of spin label to one component (Gaponenko et al 2002 J. Biomol. NMR )

35 Double half-filtered NOESY (Otting & Wüthrich 1990 Q. Rev. Biophys ) 1 H 1 1 t1 τ mix 2 2 Acq X ψ 1 ψ 2 Dec ψ = x: Hy - Iy ψ = -x: Hy + Iy A B C D ψ 1 ψ 2 x x ψ 1 ψ 2 -x x ψ 1 ψ 2 x -x ψ 1 ψ 2 -x -x Mixed dimer sample: 12 C -> 12 C 12 C -> 13 C 13 C -> 12 C 13 C -> 13 C Intramolecular: 12 C -> 12 C and 13 C -> 13 C Intermolecular: 12 C -> 13 C and 13 C -> 12 C Combination sub-spectrum ω1/ω2 (A+B) + (C+D) ω1-filter,ω2-filter unlabelled/unlabelled (A+B) - (C+D) ω1-filter,ω2-edit unlabelled/labelled (A-B) + (C-D) ω1-edit,ω2-filter labelled/unlabelled (A-B) - (C-D) ω1-edit,ω2-edit labelled/labelled

36 Double half-filtered NOESY (Folkers et al 1993 JACS ) 1 H 1 1 t1 τ mix 2 2 Acq X ψ 1 ψ 2 Dec ψ = x: Hy - Iy ψ = -x: Hy + Iy A B C D ψ 1 ψ 2 x x ψ 1 ψ 2 -x x ψ 1 ψ 2 x -x ψ 1 ψ 2 -x -x 12 C -> 12 C 12 C -> 13 C 13 C -> 12 C 13 C -> 13 C Intramolecular: 12 C -> 12 C and 13 C -> 13 C Intermolecular: 12 C -> 13 C and 13 C -> 12 C Combination (A+D) (A-D) 12 C -> 12 C and 13 C -> 13 C 12 C -> 13 C and 13 C -> 12 C

37 Structure calculation treat all distance restraints as ambiguous (contribution from each monomer, intra- or inter-monomer) include non-crystallographic symmetry (NCS) restraints to keep monomers superimposable include distance symmetry restraints to keep the interactions between monomers symmetric A 1 B 2 NCS A 1 -B 1 = A 2 -B 2 B 1 A 2 Distance symmetry A 1 -B 2 = A 2 -B 1

38 Heterochromatin protein 1 shadow chromo domain/caf peptide complex HP1 structural component of heterochromatin Binds histone H3 methylated at Lys-9, via N-terminal chromo domain Dimerizes via its C-terminal shadow chromo domain Shadow domain homodimer of 2 x 70 residues Complex with 29 residue peptide from CAF - stoichiometry is 1:2

39 N HP1 Backbone Assignment N identical in both monomers doubled; not assigned into A/B monomers

40 HP1 Backbone Assignment with NOE Information identical in both monomers doubled; not assigned into A / B monomers assigned into A and B monomers

41 Structure calculation strategy With NCS restraints only on unsplit residues, one monomer was always better definedall distance restraints being assigned to one monomer chemical shift dispersion between monomers not high enough one monomer gave better spectra NCS restraints weighted according to chemical shift degeneracy: 4 groups All peaks identical (2.0) NH only split (1.0) NH and HA split (0.5) NH and all of sidechain split (0.1) Decreasing weight Distance symmetry restraints only applied for residues that were identical