EMBO Global Exchange course, IHEP, Beijing April 28 - May 5, 2011 Introduction solution NMR Alexandre Bonvin Bijvoet Center for Biomolecular Research with thanks to Dr. Klaartje Houben 2 NMR ‘journey ’ • Why use NMR for structural biology...? • The very basics • Multidimensional NMR • Resonance assignment • Structural parameters • NMR relaxation & dynamics 3 Topics Why use NMR.... ?
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EMBO Global Exchange course, IHEP, Beijing April 28 - May 5, 2011
Introduction solution NMR
Alexandre Bonvin
Bijvoet Center for Biomolecular Research
with thanks to Dr. Klaartje Houben
2
NMR ‘journey ’
• Why use NMR for structural biology...?
• The very basics
• Multidimensional NMR
• Resonance assignment
• Structural parameters
• NMR relaxation & dynamics
3
Topics
Why use NMR.... ?
NMR & Structural biology
Dynamic activation of an allosteric regulatory protein Tzeng S-R & Kalodimos CG Nature (2009)
S2 (DS2) are unevenly distributed throughout the protein structure.More specifically, whereas most of the residues in DBD of eitherWT-CAP or CAP-S62F becomemore rigid on DNA binding, the majorityof the residues that makes up the cAMP-binding site exhibit signifi-cantly lower S2 values in CAP-S62F, indicating an enhancement inmotional freedom. It should be noted that, as indicated by chemicalshift analysis, DNA binding to CAP-S62F-cAMP2 reorganizes thecAMP-binding pocket (Supplementary Fig. 4b). The relaxation datashow that this reorganization results in enhanced flexibility of thecAMP-binding region, presumably by altering the local packingdensity21.
Order parameters values are indicative of the amplitude of spatialfluctuations experienced by a bond vector and, thus, can be related toconformational entropy19. Despite certain assumptions and limita-tions, this approach can provide reasonably accurate per-residueentropies22–27. By converting order parameters to conformationalentropy, –TDSconf, we estimate that DNA binding to WT-CAP-cAMP2 is accompanied by an unfavourable conformational entropychange (–TDSconf ,39 kcalmol21), whereas DNA binding to CAP-S62F-cAMP2 is accompanied by a favourable conformationalentropy change amounting to –TDSconf ,–22 kcalmol21 (Fig. 2b
and Supplementary Fig. 13). We conclude that the calorimetricallymeasured large entropy that drives the strong binding of CAP-S62F-cAMP2 to DNA is dominated by the favourable conformationalentropy change (Fig. 2a).
In the case ofDNAbinding toCAP-S62F-cAMP2 the calorimetricallydetermined energetics is the sum of two events: the first is the directbinding interaction between DNA and the protein molecules in theactive conformation, and the second is the population shift from theinactive to the active conformation (Fig. 4). The thermodynamics ofthe allosteric transition that results in activation can be extracted byusing CAP*-G141S, a constitutively active mutant9,28 (SupplementaryFig. 14), wherein DBD, as NMR spectra indeed demonstrate (Sup-plementary Fig. 14a), adopts largely the active conformation in theabsence of cAMP. cAMP binds to CAP*-G141S with two orders ofmagnitude higher affinity than to WT-CAP protein (SupplementaryFig. 14b). This energy difference corresponds to the energy spent bycAMP binding to elicit the active conformation to the wild-typeprotein. The combined data (Supplementary Fig. 14a–c) indicate thatthe allosteric transition of DBD from the inactive to the active con-formation requires ,3.0 kcalmol21 (DGactiv), with the process beingentropy driven (–TDSactiv5 –2.2 kcalmol21) but enthalpically opposed
Figure 2 | Energetics of CAP interaction with DNA. a, ITC bindingisotherms of the calorimetric titration of a specific DNA sequence to WT-CAP-cAMP2 (blue) and CAP-S62F-cAMP2 (magenta) and the associatedthermodynamic components (DG,DH andDS) displayed as bars. –TDSconf isthe conformational entropy as measured by NMR. b, Effect of DNA bindingon N-H bond order parameters of CAP. Changes in order parameters, DS2,for WT-CAP-cAMP2 (left) and CAP-S62F-cAMP2 (right) on DNA binding.
DS2 is given as S2 (afterDNAbinding) – S2 (beforeDNAbinding), so positiveDS2 values denote enhanced rigidity of the protein backbone on DNAbinding. The conformational entropy of DNA complex formation estimatedthrough DS2 values is unfavourable for WT-CAP-cAMP2(–TDSconf5 39.2 kcalmol21) and favourable for CAP-S62F-cAMP2(–TDSconf5 –22.3 kcalmol21). S2 plots for all WT-CAP and CAP-S62Fliganded states, including error bars, are provided in Supplementary Fig. 13.
use distinct thermodynamic strategies to interact strongly and specifi-cally with DNA.
To better understand the mechanism by which CAP-S62F-cAMP2manages to bind strongly to DNA while adopting the DNA-bindinginactive conformation, we performed a series of relaxation dispersionexperiments (Fig. 3a). These experiments have the capacity to detectand characterize low-populated conformations15,16. The results showthat on binding of cAMP to CAP-S62F, DBD resonances becomebroader, indicating the presence of exchange between conformationson the micro-to-millisecond (ms–ms) time scale. Data fitting (seeMethods) is indicative of a two-site exchange process, with the popu-lation of the excited state being ,2% (Fig. 3a). The additional linebroadening of NMR signals (Rex; Fig. 3c) caused by conformationalexchange between the ground (A) and an excited state (B) dependson the relative populations of the exchanging species (pA and pB) andthe chemical shift difference between the exchanging species(Dv)15,16. The absolute 15N Dv values of DBD residues measuredbetween the apo-CAP andWT-CAP-cAMP2 (Figs 1b and 3b) clearlycorrelate with the Dv values between the major and the minor con-formations of CAP-S62F-cAMP2 determined by relaxation disper-sion measurements (Dvdisp; Fig. 3d). Thus, the data provide strongevidence that the excited state that DBD transiently populates inCAP-S62F-cAMP2 closely resembles the active, DNA-binding com-patible conformation. Because the affinity of the active DBD con-formation for DNA (for example, in CAP-cAMP2) is many orders ofmagnitude higher than that of the inactive DBD conformation (forexample in apo-CAP), DNA will preferentially bind to the active
DBD conformation of CAP-S62F-cAMP2, despite being so poorlypopulated. Thus, the data indicate that DNA binding to CAP-S62F-cAMP2 proceeds with a population-shift mechanism17.
Despite adopting predominantly the inactive conformation andonly very poorly the active one (,2%), CAP-S62F-cAMP2 binds toDNA as tightly as WT-CAP-cAMP2, driven by a large favourablebinding entropy change, as measured experimentally by calorimetry(Fig. 2a). The amount of surface that becomes buried on binding ofDNA to WT-CAP-cAMP2 and CAP-S62F-cAMP2 is very similar,indicating that the hydrophobic effect is not the source of the largeentropy difference measured for the formation of the twoDNA com-plexes. To understand the origin of this large favourable change inentropy, we sought to determine the role of dynamics in the bindingprocess. To assess the contribution of protein motions to the con-formational entropy of the system18,19, we measured changes in N-Hbond order parameters for DNA binding to WT-CAP-cAMP2 andCAP-S62F-cAMP2 (Supplementary Figs 9–13). The order parameter,S2, is a measure of the amplitude of internal motions on the ps–nstimescale and may vary from S25 1, for a bond vector having nointernal motion, to S25 0, for a bond vector rapidly sampling mul-tiple orientations20.
DNA binding to WT-CAP-cAMP2 results in widespread increasein S2, indicating a global rigidification of the protein (Fig. 2b andSupplementary Fig. 13c). Notably, DNA binding to CAP-S62F-cAMP2 causes a large number of residues to increase their motionsas evidenced by the corresponding decrease in their S2 values (Fig. 2band Supplementary Fig. 13c). It is of interest to note that changes in
a
0.0
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(p.p
.m.)
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apo-CAP CAP-cAMP2 CAP-cAMP2-DNA
CBD
DBD
F helices F helices
b c
Figure 1 | Conformational states of CAP and effect of cAMP bindingassessed by NMR. a, Structures of CAP in three ligation states: apo9,cAMP2-bound
10, and cAMP2-DNA-bound8. The CBD, DBD and hinge
region are coloured blue, magenta and yellow, respectively. cAMP and DNAare displayed as grey and green sticks, respectively. b, c, Effect of cAMP
binding on the structure of WT-CAP (b) and CAP-S62F (c) as assessed bychemical shift mapping (Supplementary Fig. S4). Chemical shift difference(Dv; p.p.m.) values are mapped by continuous-scale colour onto the WT-CAP-cAMP2 structure.