Post on 06-Feb-2018
Protein-proteininteractions by NMR
Slow
Fast kon,off >> (νfree - νbound )
νfree νbound
A + B ABkon
koff
kon,off ~ (νfree - νbound )
kon,off << (νfree - νbound)
Measure Kd by e.g. fluorescence, ITC, Biacore, NMR
Minimize interacting region, especially of peptidese.g. limited proteolysis
Find conditions where both components are stableand 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)
strong
strong
weak or strong
weak or strong
weak or strong
Structures of protein complexes
Dynamics of interactions
Mapping interactions e.g. for mutagenesis studies
Extracting distance information
Docking
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
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 transferringthem by chemical exchange to the free state, where they can beobserved
Requirement is that off-rate is fast: the ligand must associate/dissociate afew times during the mixing time
Transferred NOEs are larger than free intra-ligand NOEs and havethe same sign as diagonal peaks
Intermolecular NOEs are low intensity due to low protein concentration
Clore & Gronenborn: J. Mag. Res. 48 402-417; J. Mag. Res. 53 423-442Ni & Scheraga Acc. Chem. Res. 27 257-264 (review)
Studying strong interactions: structures of complexes
2 types of experiment:Edited or separated: keep 1H attached to nucleus XFiltered or rejected: keep 1H NOT attached to nucleus X
Differential Labelling
13C,15N-proteinUnlabelled peptide 13C-edited NOESY
NOEs between 13C-1H and all other 1H (13C-1H, 12C-1H, 15N-1H, 14N-1H)
X-filtered, 13C-edited NOESYNOEs between 13C-1H and 12C-1H/15N-1H
Double-filtered NOESYNOEs between 12C-1H and 12C-1H
H
H
13C
13C
12C
H
12C
H
14N
H
15N
H
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
Tight Complexes: Slow Exchange
Chemical Shift Assignments
1. Backbone experiments:HSQC, HNCA, HN(CO)CA, HNCACB, CBCA(CO)NHrecorded on each component of complex
2. Sidechain experiments:15N-separated TOCSY, HCCH-TOCSYCC(CO)NH , H(CCCO)NH, HBHA(CBCA)(CO)NHon each component of the complex
3. Coupling constant experiments:HNHA on each component of the complex
Tight Complexes: Slow Exchange
NOE Assignments
4. Intra- and inter-molecular NOEs mixed:3D 13C-separated NOESY3D 15N-separated NOESY
5. Intermolecular NOEs:X-filtered experiments. 3D 13C-separated if sensitivity is good or 2D 1H/1H version
(Zhwalen et al JACS 1997 119 6711-6721)
X-filtered Experiments
Two basic types:
a) Schemes where X-attached 1H are removed directly
1/2J1H
e.g. generate MQCX
b) Schemes where interleaved experiments are recordedand have to be added or subtracted to keep or discard X-attached 1H
X-pulse present or absent inalternating experiments
1H
X
X-filter (I)Purge sequence
1H ∆1 ∆2 I = 1H attached to X nucleusH = all other 1Hy
XG2G1 G1
G
Hz + Iz -Hy - Iy cosπJIS∆1 + 2IxSzsinπJIS∆1
(∆1 = 1/2JIS) -Hy + 2IxSz-Hy - 2IxSy
90˚(1H), ∆1
90˚(X)
1. ∆1 and ∆2 can be tuned for different values of JIS e.g. 120-145 Hz (aliphatic) and 160-220 Hz (aromatic) 2. Can add spin-lock (y) on 1H to remove Ix-based antiphase that escapes X pulses3. Replaces a 1H 90˚ pulse in a sequence
X-filter (II)Refocussed half filter
Hz + Iz -Hy - Iy -Hy -IycosπJIS∆1 + 2IxSzsinπJIS∆1 (∆1 = 1/2JIS)Hy - 2IxSz Hy - Iy
OR Hy + 2IxSz Hy + Iy
1H ∆1 ∆1
X
GG1 G1
I = 1H attached to X nucleusH = all other 1H
90˚(1H)
180˚(1H,X)∆1
∆1
180˚(1H) ∆1
AB
A + B = Hy only (filtered)A - B = Iy only (edited)
All 13C-filtered experiments suffer because 1JCH varies:120-145 Hz aliphatic CH; 160-220 Hz aromatic CH∆1 delays are inevitably only tuned to one J coupling
220
200
180
160
140
12020 40 60 80 100 120
1JCH (Hz)
aliphatic
Tyr, Phe, Trp
His
δ13C (ppm)
1JCH = (0.365 ± 0.01 Hz/ppm) δC + 120.0 ± 0.5 Hz
Zwahlen et al (1997) JACS 119 6711-6721
Adiabatic pulses
Trajectory of 13C magnetization follows effective field for duration of the pulse
Carrier frequency of the pulse starts far upfield, sweeps through resonance, then
downfield
13C 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
X-filter (III)Purge scheme with adiabatic pulse
Hy + Iy Hy + IycosπJIS(∆1-2t) - 2IxSzsinπJIS(∆1-2t)Hz + IzcosπJIS(∆1-2t) - 2IxSzsinπJIS(∆1-2t)
1H
X
G
∆1/2 ∆1/2-t
G1 G1
t
G2
a b 13C spin inverted at time t (t≥0) after 1H 180˚ pulse 1JCH evolution occurs for a time ∆1-2t1JCH and t vary for each 13C nucleus
From a to b:
removed by G2IzcosπJIS(∆1-2t) = 0 for all spins∆1-2t = 1/(21JCH) (i)
Since t≥0 and 1JCH is smallest for upfield 13C (i.e. methyls), these must be inverted first. ∆1 is set close to 1/2 1JCH (methyl)
1JCH = AδC + B (ii) (A=0.365 Hz/ppm; B = 120.0 Hz)
Frequency of the transmitter δRF(t) = δC. Substitute for 1JCH in (i)
∆1-2t = 1/[2(AδRF + B)] time derivative to get sweep rate during WURST
3D 13C-filtered, 13C-edited NOESY-HSQC
Two of these modules at the start of the sequence (shorter than the original half-filter)
Suppression factors are 100-140-fold
Putting filter modules at the start of the sequence makes H2O suppression easier
Filters add to the length of the sequence, so semi-CT, concatenating 1H chemical shift evolution with the half filter
15N filter at the same time as the 13C filter
Cdc42 - 21 kDa small G protein of the Rho familyGTP cofactor replaced by GMPPNP
PAK - 5kDa fragment - can be expressed as GST-fusion
Kd ~ 30nM
Samples:
15N Cdc42 + unlabelled PAK15N,13C Cdc42 + unlabelled PAK15N PAK + unlabelled Cdc4215N,13C PAK + unlabelled Cdc42
no deuteration required - all experiments worked
3D X-filtered, 13C-edited NOESY ran on 13C,15N PAK, unlabelled Cdc42
Summary of Cdc42/PAK Intermolecular NOEs
Unambiguous Ambiguous
13C-filter/13C-edited 32 2913C-NOESY (PAK) 67 7313C-NOESY (Cdc42) 21 40
15N-NOESY (PAK) 19 515N-NOESY (Cdc42) 7 12
Total Intermolecular 146 159
Total number of NOEs = 4,000
Assignment of Unlabelled Component in Complex
Filtered/rejected experiments:to assign unlabelled peptide with labelled protein- sensitivity/water suppression poordouble filter (ω1 and ω2) required in NOESY but not inthrough-bond experiments
J(CH,NH)-separated NOESY -- better sensitivity and water behaviour(Nietlispach)
Double half-filtered NOESY(Otting & Wüthrich 1990 Q. Rev. Biophys. 23 39-96)
∆1 ∆1 t1 τmix ∆2 ∆2
Decψ1 ψ2
1H Acqψ = x: Hy - Iyψ = -x: Hy + IyX
12C -> 12C
12C -> 13C
13C -> 12C
13C -> 13C
Aψ1 ψ2x x
Bψ1 ψ2-x x
Cψ1 ψ2x -x
Dψ1 ψ2-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
J(CH,NH)-separated NOESY(based on Melacini, JACS 122 9735-9738)
After the semi-CT period, required magnetization is:-Iy cosπJt2 cosΩHt1 modulated by J in t213C-bound 1H are at ±JCH/2; 12C-bound 1H are at J=0
J=0 plane contains intra-peptide NOEs and peptide(f1) to protein (f3) NOEsIntra-protein NOEs and protein (f1) to peptide (f3) NOEs are at ±JCH/2
Intermolecular NOEs in J=0 plane are coupled in F3Intra-peptide NOEs have a return peak in the same plane
Chromodomain complex with methylated peptide from histone H3
Problematic assignment of Me2-Lys - not J coupled to anything else
Assignment of NOEs from Me2-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 Me2-Lys)
xF3:J =0 Hz
F3:J =160 Hz
Me-K9Trp 42Phe 45
Trp 42
Phe 45
Me-K9
160Hz
1H [ppm]
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 13C- and 15N-edited spectra: check that they do not have any inter-molecular possibility (they may not appear in X-filter, 13C-edited experiment if they are weak)
Ambiguous NOEs that appear in 13C-edited NOESY as well as X-filter, 13C-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. 13C-edited NOESY: F1/F3 = 13CH only; F2 = any 1H
SH3/peptide Calculation Strategy
•All intermolecular distance restraints were ambiguous (32 from 13C-filtered/13C-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
NOEs used in SH3/peptide Structure Calculations
Unambiguous Ambiguous
13C-filter 0 3213C-NOESY (SH3) 1026 55415N-NOESY (SH3) 395 1231H NOESYs 85 29
Totals 1506 738
At end of calculation - 66 intermolecular NOEs
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
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 15N and 1H shifts, often defined as:[(∆15N)2 x 10(∆1H)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.
Using Chemical Shift Mapping Data for Docking(HADDOCK)
Take significant shift changes, screened by solvent accessibilityActive residues
Take residues close to active residues on the surfacePassive residues
Ambiguous interactive restraint (AIR)
Natoms NresB Natoms
diAB = Σ Σ ΣmiA=1 k=1 nkB=1
between any atom m of active residue i in protein A (miA) and any atom n ofboth active and passive residues k (Nres total) of protein B (nkB) and inverselyfor 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.
1d6miAnkB
( )(-1/6)
Dominguez et al (2003) J Am Chem Soc 125 1731-1737
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 (Eelec, EvdW, EAIR) andaverage buried surface area
Lowest energy cluster with the highest buried surface area assumedto be correct
Can also add RDCs, inter-molecular NOEs and radius of gyration term to prevent expansion at the interface (Clore(2003) JACS 125 2902-12)
Cross-saturationTakahashi et al (2000) Nat. Struc. Biol. 7 220-223
-NH
-CH
-15NH
-C2H
ProteinI
ProteinII Strong binding case
R.F.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 H2O, it does not affect amides of protein I.
Cross-saturation to the 15NH 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
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 labelledaddition of spin label to one component (Gaponenko et al 2002 J. Biomol. NMR 24 143-148)
Double half-filtered NOESY(Otting & Wüthrich 1990 Q. Rev. Biophys. 23 39-96)
∆1 ∆1 t1 τmix ∆2 ∆2
Decψ1 ψ2
1H Acqψ = x: Hy - Iyψ = -x: Hy + IyX
12C -> 12C
12C -> 13C
13C -> 12C
13C -> 13C
Aψ1 ψ2x x
Bψ1 ψ2-x x
Cψ1 ψ2x -x
Dψ1 ψ2-x -x
++ +
+++
--
--
++++ --
Mixed dimer sample:
Intramolecular: 12C -> 12C and 13C -> 13CIntermolecular: 12C -> 13C and 13C -> 12C
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
Double half-filtered NOESY(Folkers et al 1993 JACS 115 3798-3799)
∆1 ∆1 t1 τmix ∆2 ∆2
Decψ1 ψ2
1H Acqψ = x: Hy - Iyψ = -x: Hy + IyX
12C -> 12C
12C -> 13C
13C -> 12C
13C -> 13C
Aψ1 ψ2x x
Bψ1 ψ2-x x
Cψ1 ψ2x -x
Dψ1 ψ2-x -x
++ +
+++
--
--
++++ --
Intramolecular: 12C -> 12C and 13C -> 13CIntermolecular: 12C -> 13C and 13C -> 12C
Combination(A+D) 12C -> 12C and 13C -> 13C(A-D) 12C -> 13C and 13C -> 12C
Structure calculationtreat 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
A1
B1
B2
A2
NCS A1-B1 = A2-B2
Distance symmetry A1-B2 = A2-B1
Heterochromatin protein 1shadow 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
HP1 Backbone Assignment
identical in both monomers
doubled; not assigned into A/B monomers
N N
HP1 Backbone Assignment with NOE Information
112 115
120
127133
138145
147 149
155
158
166
doubled; not assigned into A / B monomers
assigned into A and B monomers
identical in both monomers
Structure calculation strategy
With NCS restraints only on unsplit residues, one monomerwas always better defined-
all distance restraints being assigned to one monomerchemical shift dispersion between monomers not high enoughone monomer gave better spectra
NCS restraints weighted according to chemical shift degeneracy:
4 groupsAll peaks identical (2.0)NH only split (1.0)NH and HA split (0.5)NH and all of sidechain split (0.1)
Distance symmetry restraints only applied for residuesthat were identical
Decreasingweight