Protein-protein interactions (PPIs) via NMR Paola Turano turano@cerm.unifi.it
The magnetic field at the The chemical shift nucleus (the effective field) is generally less than the applied field by a fraction s: B = B 0 (1-s) Simple shielding effects: eletronegativity more electron withdrawing-- less shielded N H shielding cones shielding deshielding deshielding shielding
1 H (ppm) MORE SHIELDED
Internal protein dynamics Considering the rotational correlation time of the protein in solution taken as a rigid body as a pivotal point of the timescale, we can define as internal fast motions those faster than the tumbling correlation time and as collective conformational equilibria those involving processes slower than the tumbling correlation time.
Exchange process: a dynamic process that exposes a nucleus to at least two distinct chemical environments Two-site exchange I x P x obs = f A A + f B B = 0.75 A + 0.25 B
The exchange regime is determined by the chemical shift separation (in Hz). Can be modulated by T (affecting k ex )and B 0 (affecting ).
Reaction scheme: Special case: ligand binding to a protein The exchanging sites are the free (P) and the complexed form (PL) of the protein. The exchange rate is given by:
Line shapes simulated for the one-step binding mechanism for increasing populations of the complex (from blue to red). = 250 Hz k ex = 2000 Hz.
Thermodynamics of ligand binding P + L PL K a = [PL]/[P][C] PL P + L K d = [P][L]/[PL] Protein-protein interactions (PPIs) span an affinity range that is extremely broad, with dissociation constants K d from 10-2 to 10-16 M.
Affinity constant and dissociation constant K d = [P][L]/[PL] Strong PPIs: Weak PPIs: Ultra weak PPIs: dissociation constant K d < 1 M 1 M < K d < 100 M K d > 100 M
Kinetics In the assumption of one-step reaction: PL k off k on P+ L K d = k off k on Different combinations of k off and k on can give rise to similar affinities.
k ex, k on, k off The NMR exchange rate is given by: What are the factors affecting k on and k off in PPIs?
Association rate constant k on Wide spectrum of measured association rate constants. The red vertical line marks the start of the diffusion-controlled regime. The shaded range marks the absence of longrange forces.
Association rate constant k on The association of two proteins is bounded by the rate at which they find each other through diffusion. To form a stereospecific complex, the two molecules must have appropriate relative orientations when they come together. The rate of association of a protein complex is limited by diffusion and geometric constraints of the binding sites and may be further reduced by subsequent chemical processes.
Association rate constant k on A diffusion-controlled rate constant falls on the high end of the spectrum of observed values. A reaction-controlled rate constant falls on the low end A diffusion-controlled association typically involves only local conformational changes between the unbound proteins and the native complex. A reaction-controlled association typically involves gross changes such as loop reorganization or domain movement.
Association rate constant k on : electrostatic contribution To go beyond the basal rate constant k D0 and reach values > 10 7 M -1 s -1, as observed for many protein complexes, intermolecular forces must be present. For protein-protein association the dominant long-range force is provided by electrostatic interactions.
Association rate constant k on For protein-protein association the dominant long-range force is provided by electrostatic interactions, as manifested by complementary charge distributions on the binding partners, which are illustrated in the figure for four protein pairs.
Kinetics vs. thermodynamics When several proteins compete for the same receptor or when one protein is faced with alternative pathways, kinetic control, not thermodynamic control, dominates in many cases; this is especially true when dissociation is slow. Differences in binding rate between related proteins may serve as an additional mechanism for specificity.
Kinetics vs. thermodynamics When several proteins compete for the same receptor or when one protein is faced with alternative pathways, kinetic control, not thermodynamic control, dominates in many cases; this is especially true when dissociation is slow. Differences in binding rate between related proteins may serve as an additional mechanism for specificity.
Dissociation rate constant k off Dissociation is a first order reaction whose rate is dictated by the strength of short range interactions between the interacting proteins (van der Waals interactions, hydrogen bonds, hydrophobic interactions and salt bridges) Typical k off are in the 10 4-10 -7 s -1 range.
Dissociation rate constant k off k off is directly related to the lifetime of the complex: mean lifetime = 1/k off half-life = ln2/k off A higher value of k off means a shorter lifetime for the complex and vice versa.
Transient complexes Transient complexes form when a high turnover is a functional requirement and their components associate and dissociate rapidly, namely with k off 10 3 s -1 and k on in the range of 10 7-10 9 M -1 s -1. This results in dissociation constants typical for weak and ultra weak complexes and lifetimes ms. Revealing the presence of such interactions is experimentally challenging because they do not result in a sufficient amount of complexes that can be directly detected.
k off in electrostatic interactions The association rate constant decreases significantly with increasing ionic strength, whereas the dissociation rate constant is only modestly affected by ionic strenght. As the transient complex lies at the outer boundary of the interaction energy well and hence is close to the native complex, ionic strength is expected to screen electrostatic interactions in the two types of complexes to nearly the same extent. Hence, the association constant and association rate constant are expected to have nearly the same dependence on ionic strength and the dissociation rate would be little affected by ionic strength. K a = k on k off
Association rate constant k on Electrostatic interactions. Rates beyond the 10 5 10 6 M -1 s -1 range implicate electrostatic enhancement. A hallmark of electrostatically enhanced diffusion-limited association is manifested by disparate ionic-strength effects on the association rate k on and the dissociation rate k off. Specifically, k on decreases significantly with increasing ionic strength, but k off is affected by ionic strength only marginally
Affinity constant and dissociation constant K d = [A][B]/[C] Strong PPIs: Weak PPIs: dissociation constant K d < 1 M 1 M < K d < 100 M Ultra weak PPIs: K d > 100 M NMR = unique tool to monitor these interactions
NMR can be used to study protein ligand interactions, by acquiring multiple NMR spectra along a [P]/[L] titration coordinate. As a rule of thumb, in the diffusion controlled association regime: Kd nm Kd M Kd mm
1 H- 15 N HSQC = gold standard for monitoring intermolecular interactions The 1 H- 15 N HSQC spectrum is a signature of the protein structure
The 1 H- 15 N HSQC experiment folded 1 H chemical shift as a signature of the degree of protein folding Partially folded unfolded
1 H- 15 N HSQC in interaction studies THE CHEMICAL SHIFT OR AMIDES OF RESIDUES IN THE INTERACTION AREA IS EXPECTED TO CHANGE UPON COMPLEX FORMATION DUE TO THE CHANGE IN THE CHEMICAL ENVIRONMENT OF THE NUCLEUS. 1 H NUCLEI ARE VERY SENSITIVE REPORTES OF CHANGES IN THEIR ENVIRONMENT Different strategies for fast, slow and intermediate exchange
1 H- 15 N HSQC: fast chemical exchange
1 H- 15 N HSQC: fast chemical exchange obs = f free free + f bound bound Measurable effects on obs even in the case of ultra weak interactions (up to mm K d s) All the other NMR observables are also population-weighted, including relaxation rates.
Chemical shift perturbation (CSP) mapping via HSQC 1 H- 15 N HSQC = simple and fast experiment, with cryoprobe technology can be acquired in a few minutes even for very low protein concentrations (down to tens of M). In interaction experiments, the 1 H- 15 N HSQC spectrum of one protein is monitored when the unlabeled interaction partner is titrated in and the chemical shift perturbations are recorded for each amino acid. Provided the assignment of the 1 H- 15 N HSQC spectrum is available, shift perturbation measurements identify residues at the interface. If a structural model of the protein exists residues undergoing meaningful chemical shift perturbations are mapped on the protein structure, thus providing the location of the interface. In the case of protein-protein interactions, the procedure is repeated for the second partner.
CSP mapping: fast exchange Identification of residues undergoing chemical shift perturbations follwoing them in a titration Garrett plot 1 H- 15 N HSQC Δ av =[(Δδ 2 NH+(Δδ N /5) 2 )/2] 1/2 Mapping of the chemical shift perturbations on the protein surface. Data driven docking to define a structural model Bertini, Chevance, Del Conte, Lalli, Turano PLoS One. 2011 6(4):e18329. Contact surface areas are identified if assignment is available for both partner macromolecules
Data Driven Docking Chemical shift perturbation mapping yields the interaction area on the individual binding partners, but does not allow defining the relative orientation of the two molecules nor the atom-to-atom interactions at the basis of the recognition process. Nevertheless, residues undergoing chemical shift perturbations can be used as selection filters in data driven computational soft-docking programs
K d measurements Under fast exchange conditions: Simultaneous fit of the chemical shift changes K d = [A][B]/[C] K d values were obtained by plotting the weighted average chemical shift variations of perturbed residues on the 15 N- enriched Bcl-x L as a function of the concentration of the unlabeled partner cytochrome c and were found to be in the 1-3 mm range.
Optimal K d /[B] when titrating with A Protein in excess of 10-50*Kd results in very large errors in measurement [B] >> K d Lack of curvature makes the fitting difficult/impossible [B] << K d Small amounts of protein with weak binding require a large excess of ligand to achieve saturation Fitting is good but too much ligand is needed Curve is good when [B] K d
Chemical shift mapping: limitations of the approach In the presence of binding-induced conformational rearrangements, the chemical shift perturbation may extend to residues that are not at the interface, and the chemical shift perturbation fails as a mapping tool, although it still represents an excellent indicator of allosteric processes. Such a situation is associated to slow k on.
1 H- 15 N HSQC: intermediate exchange regime During the titration signals may be broadened beyond detection due to exchange broadening and can be retrieved at the end (if we can get the final adduct!).
Chemical shift mapping under slow exchange conditions Structural info can be derived only if we know the assignment The problem of signal assignment
EXSY experiments: a possible tool for the (straightforward) assignment under slow exchange regime Homonuclear 1 H- 1 H or 13 C- 13 C-like experiments EXchange SpectroscopY (EXSY), also known as the zz-exchange experiment. τ ex 10 5000 ms; k ex 0.2 100 s -1 Observing signals of the bound and free forms in the same NMR spectrum is a prerequisite.
Slow exchange If the exchange rate is << R 2, then the exchange event has little or no effect on the linewidth. But, if the exchange rate is > R 1 it may be possible to measure the rate constants by detecting the exchange of magnetization between the nuclei in the two environments. EXchange SpectroscopY (EXSY), also known as the zz-exchange experiment. τ ex 10 5000 ms; k ex 0.2 100 s -1
(a) EXSY experiments heteronulcear (b) (c) Slow exchange with respect to the chemical shift time scale, but fast with respect to T 1 Here m is the mixing time!!!
Other NMR observables for PPIs Changes in protein dynamics. Changes in the overall tumbling time. Distance and orientational restraints: NOE RDC Paramagnetic effects
NMR for the characterization of PPIs: other tools Changes in the overall tumbling time: establishment of intermolecular interactions gives rise to changes in the size of the system under study. For protein-protein interactions an overall increase in 15 N transverse relaxation rate values is observed, which is consistent with an increase in the overall tumbling correlation time upon complex formation. In the fast exchange regime, the measured transverse relaxation rate is an average of the transverse relaxation rates of the free and bound protein forms, weighted by their molar fraction. This phenomenon can be used to establish complex formation, but also causes signal broadening in the spectra of the bound form of a protein, thus often requiring ad hoc experimental procedures for the obtainment of 1 H- 15 N correlations at the basis of the chemical shift perturbation mapping approach, as detailed below. Under slow exchange signals may become undetectable in HSQC TROSY, CRIPT/CRINEPT TROSY: monolateral chemical shift mapping for complexes up to 1 MDa,
Changes in protein dynamics Interaction between two molecules not only implies changes in chemical environment due to local/extended conformational variations but also affects the solvent accessibility and internal protein mobility. Reduced solvent accessibility of the contact area with exclusion of bulk water. Some solvent molecules may remain at the interface: bridging; non-bridging; simply trapped. Changes in internal dynamics. The side chain (exemplified by an arrow) of a residue of the blue protein in its free form exists in a number of energetically similar conformations, that can be represented by a dynamic ensemble of interconverting states. Upon binding the side chain assumes a single conformation
Changes in protein dynamics Changes in backbone dynamics MMP Rigified: blue Unaffected : green Faster: red TIMP-1 J. Mol. Biol. (2003) 327, 719 734
Changes in the overall tumbling time Establishment of intermolecular interactions gives rise to changes in the size of the system under study. For protein-protein interactions an overall increase in 15 N transverse relaxation rate values is observed, which is consistent with an increase in the overall tumbling correlation time upon complex formation.
log (T 1,2 ) Molecular tumbling: r r is larger for larger molecules Stokes Einstein Debye equation, where the molecule is considered as a rigid sphere: r = 4phr 3 /3kT = hmw/dn A kt h= viscosity r = radius of the molecule (assumed to be spherical) d = density of the molecule ( 10 3 kg m -3 ) T 2-1 is larger for larger molecules NMR linewidth is larger for larger molecules -log ( r ) T 2-1 = p 1/2
Changes in the overall tumbling time Under the fast exchange regime, the measured transverse relaxation rate is an average of the transverse relaxation rates of the free and bound protein forms, weighted by their molar fraction. This phenomenon can be used to establish complex formation, but also causes signal broadening in the spectra of the bound form of a protein, thus often requiring ad hoc experimental procedures for the obtainment of 1 H- 15 N correlations at the basis of the chemical shift perturbation mapping approach Under slow exchange signals may become undetectable in HSQC TROSY, Cript/CRINEPT TROSY): monolateral chemical shift mapping for complexes up to 1 MDa,
Line narrowing strategies: 2 H-enrichment + ad hoc experiments CRIPT transfer efficiency 400 KDa Optimal MW (kda) 30-100 800 KDa >100
Chemical shift mapping in slow-exchange HasR 98 kda in DPC micelles apohasa in HasA-HasR 19 kda HSQC vs. CRINEPT-TROSY 3 classes of signals: Not affected ( < 0.25 ppm) Disappearing from their original well-resolved position Behavior not safely defined Caillet-Saguy, Piccioli, Turano, Izadi-Pruneyre, Delepierre, Bertini, and Lecroisey, JACS 2009
HasA-HasR: interaction surface MONOLATERL INFORMATION HasA in the apo open conformation HasR model structure Caillet-Saguy, Piccioli, Turano, Izadi-Pruneyre, Delepierre, Bertini, and Lecroisey, JACS 2009
Intermolecular NOEs The gold standard for the obtainment of structural information in protein NMR is the Nuclear Overhauser Effect SpectroscopY (NOESY) experiment, in either its 2D homonuclear version or in the 15 N- or 13 C edited 3D variants, which provides 1 H- 1 H cross peaks for all pairs of protons that fall at short distance (within about 5 Å) one from the other. In a rigid system, translation of NOE intensities into distances is possible thanks to the linear correlation between peak volume and 1/r 6 (where r is the proton-proton distance). Dipolar interactions across a binding interface can, in principle, give rise to NOEs, which can thus provide intermolecular distance restraints. However, it is often difficult to determine such NOEs. In the case of a complex, the correlation time for the dipolar interaction can be dominated by the exchange time between the free and bound form. Dissociation rates faster than the tumbling are effective in reducing the dipolar interaction even down to values where the NOE effect is no more measurable. In practice intermolecular NOEs to define the three-dimensional structure of a proteinprotein complex are essentially restricted to tight complexes (K d <10 M). Under these conditions, isotope-edited and -filtered NMR pulse sequences are used to distinguish between inter- and intramolecular NOEs
Intermolecular NOEs may be quenched by fast chemical exchange h c /r 6 Now we are interested in measuring intermolecular distances, r. The correlation tome for the intermolecular interaction is determined by : c -1 = r -1 + ex -1 where the former is the correlation time for mulecular tumbling and the second the chemical exchange correlation time. The latter becomes dominant when ex -1 < r -1
Residual Dipolar Couplings (RDCs) RDCs provide long range structural restraints for NMR structure determination of macromolecules that are not accessible by most other NMR observables, which are dependent on close spatial proximity of nuclei The measured effect is small and specific experiments have been developed for the measurement of H-N or H-C RDC s. RDC s are effective in defining the relative orientation of pairs of nuclei (most commonly 1 H- 15 N of backbone amides) within a molecular frame. RDCs can also be used for the purpose of deriving the relative orientation of two proteins in a complex. An orienting agent is needed. Under fast exchange conditions, in order to overcome the difficulties encountered in obtaining RDCs that emanate from the complex alone, a titration approach can be employed where RDCs are measured in different equilibrium mixtures of the free and bound form. While the RDCs of the free states can be measured directly, the RDCs originating from the bound state will be obtained indirectly by extrapolation of the RDCs in the different equilibrium mixtures.
NMR for the characterization of PPIs: Paramagnetic derived constraints. Observable other tools Paramagnetic NMR observables as structural restraints Dependence upon structural parameters Iron-metal distance Contac shift no no Angles Pseudocontact shift r MH -3 MH & MH T 1,2-1 contact No no T 1,2-1 dipolar r MH -6 no T 2-1 Curie r MH -6 no Paramagnetic residual dipolar coupling no MH & MH Cross correlation btw Curie & dipolar relaxation r MH -3 CCR
Suggested readings Schreiber G, Haran G, Zhou H-X (2009) Fundamental Aspects of Protein-Protein Association Kinetics. Chem. Rev. 109:839-859. Zuiderweg ER (2002) Mapping protein-protein interactions in solution by NMR spectroscopy. Biochemistry 41:1-7. Kleckner IR, Foster MP (2011) An introduction to NMR-based approaches for measuring protein dynamics. Biochim Biophys Acta. 4:942-68. Del Conte R, Lalli D, Turano P (2013) NMR as a tool to target protein-protein interaction. In: Disruption of Protein-Protein Interfaces, Ed. Mangani S. - Springer Heidelberg New York Dordrecht London. pp.: 83-111.