Cell, Volume 139
Supplemental Data Transient Non-native Hydrogen Bonds Promote Activation of a Signaling Protein
Alexandra K. Gardino, Janice Villali, Aleksandr Kivenson, Ming Lei, Ce Feng Liu, Phillip Steindel, Elan Z. Eisenmesser, Wladimir Labeikovsky, Magnus Wolf-Watz, Michael W. Clarkson, and Dorothee Kern
Figure S1: 15N CPMG relaxation dispersion for wild-type (A), BeF3--activated (B) S85D (C), and Y101F NtrCr (D) at 800 MHz and 25C. The dispersion curves are color coded as followed: residue 5 (peach), 8 (gold), 10 (burnt sienna), 11 (black), 12 (slate), 13 (strawberry), 16 (rose), 29 (salmon), 37 (burgandy), 40 (dark gray), 49 (sage), 50 (mustard), 55 (yellow-green), 64 (brown), 66 (blue-green), 69 (red), 71 (light gray), 88 (orange), 89 (dark green), 90 (light brown), 91 (navy blue), 102 (royal blue), 106 (sea green), 107 (jungle green), 114 (tan), 122 (dark brown). Global fitting of the 600 MHz and 800 MHz CPMG data for the BeF3- activated form and the transition-state mutants (S85D and Y101F) confirmed both the exchange rates and the populations previously determined using the chemical shift changes of all mutant forms together with the CPMG experiments at 600 MHz only, therefore independently verifying the robustness of determining these kinetic parameters using the approach described in Experimental Procedures. For wild-type, such a global fit is not possible because the exchange rate is too fast to be fully suppressed by the maximal CPMG field strength. Here, exchange rates were determined according to (Gardino and Kern, 2007), and
(A) Chevron plot depicting the natural log of the observed rate (kobs).. Rates were measured using stopped-flow fluorescence at various guanidinium-hydrochloride concentrations at 25°C. 1994). where kobs is equal to the sum of the unfolding rate and folding rate (kobs= ku + kf) for WT (red ▲). According to (B) for wild-type NtrCr. For wild-type and mutants
. Error in fluorescence unfolding data are s.d. and D86N/A89T mutants do not.3 M GdmCl due to the increasing contribution of kf with an inflection point at 2..populations as described in Experimental Procedures. D86N.5M (ku = kf). from the mean. in which Rex is proportional to Δω2. Schutkowski et al.d. The CPMG data at 800 MHz are consistent with the inactive/active conformational exchange occurring in the very fast NMR time regime (kex>> Δω). (C) Kinetic scheme illustrating why the
carbamoylphosphate-activated NtrCr shows the characteristic U-shape of a chevron plot in the region of the folding transition while wild-type.
Figure S2. Rates for proline isomerization at 25°C were estimated from previously
published data (Reimer et al. Kinetic and thermodynamic data for folding of inactive and active wildtype and mutant forms of NtrCr measured by fluorescence. and CPO4-activated NtrCr (black ●). 1998. Error in relaxation rates are from the larger of the difference in duplicate points or 2% of the signal to noise. D86N (orange ■). We propose that the lack of curvature in the transition region of the Chevron plot is due to the cis/trans isomerization of the Pro 105 cis-proline in the structure of NtrCr after unfolding. kobs should curve below 3. D86N/A89T (yellow ♦). Error in unfolding rates are s. (B)
Thermodynamic unfolding curve for wild-type NtrCr measured by fluorescence after equilibration for 1 hour at 25°C at various GdmCl concentrations. from the mean.
. We note that a very similar model was used in folding studies of the homologous protein CheY (Munoz et al. Folding rates plotted with open symbols in (A) are from the double jump experiments. NMR. a double exponential process as detected with a fast rate followed by a slow rate of about 10-2 s-1. for PNtrCr the cis/trans isomerization is one order of magnitude faster than the refolding rate consequently is no longer rate limiting. (C) Change in intensity with increasing GdmHCl (same data as in a) fit to a modified sigmoidal characterized by a two-state transition centered ~2. In contrast. When refolding was initiated by dilution of the denaturant after incubation.
Figure S3. the time regime indicative for prolyl peptide bond isomerization. Rates of folding and unfolding determined from the chevron plot are listed in Table S2.
Quantitative fits of the folding/unfolding equilibrium measured by
(A) Change in peak intensity for W7 (peak of the folded state in a 15N 1H HSQC) in wildtype NtrCr with increasing denaturant concentration.4M GdmHCl. the cis/trans isomerization is at the same magnitude as the refolding rate resulting in the lack of curvature in the Chevron plot since there is no significant refolding because the majority of molecules are in the wrong isomerization state (trans). This results in the typical curving of the Chevron plot for the latter. This model is buttressed by our stopped flow refolding experiments.forms. 1992). The second slow process was eliminated in a double jump experiment in which the protein was only allowed to unfold for 10 seconds (Kiefhaber and Schmid. 1994). (B) Effect of increasing concentrations of salt (NaCl) on the peak intensity of W7 follows a single exponential.
and thus no information on the unfolding/folding transition. Error derived from the r. or overlapped. prolines.d. (D) Plot of peak intensity after subtraction of the ionic strength effect which can now be fitted to a standard twostate sigmoidal. A) and m-value (MG. Grey residues are unassigned.s. from the mean over a 1ppm (1H dimension) by 3ppm (15N dimension) in a peak free region of the individual spectra to estimate spectral noise. This lead to a complete loss of the peak intensity at small concentrations of denaturant due to the effect of increasing ionic strength of the sample. B) for each residue in NtrCr fitted as described in Fig.s. from the mean over a 1ppm (1H dimension) by 3ppm (15N dimension) in a peak free region of the individual spectra to estimate spectral noise. S3.
Figure S5. Residues that were fit as described in Fig.m.d. Residues in yellow are assigned peaks whose intensities were small due to exchange broadening. The change in free energy of unfolding (ΔGUF.corresponding to the unfolding transition. Error from the r.
. S3 to determine their change in free energy of unfolding (ΔGUF) are plotted onto the inactive state structure in red. The intensity of each peak in the absence of denaturant (C) showing that residues with large errors in ΔGUF (A) and MG (B) correspond to residues with lower peak intensities due to exchange broadening.m. and an exponential to account for the change in intensity due to an increase in ionic strength of the solution.
Rates were measured using stopped-
flow fluorescence at various guanidinium-hydrochloride concentrations at 25°C. which are listed in Table S3. (A) Top panel: Time traces of backbone root mean square deviation (RMSD) along the MD trajectories of the inactive (blue) and active (red) states. Error in unfolding rates are s. (B) Bottom panel: The backbone root mean square fluctuation (RMSF) of the simulation trajectories between 9 15 ns are plotted for wild-type inactive (thick blue line) and active (thick red line). (B) The unfolding rates (see also Fig. S 2) of wild-type (red (blue ) and S85D
) are identical within experimental error. S85D mutation that removes a non-native hydrogen bond of the transition does not affect the ground states (inactive and active state).
Figure S7. S85D and S85G support the experimental finding that the ground states are not significantly changed by these amino acid substitutions.
. Unbiased MD simulations in explicit water for wild-type. from the mean. The only small noticeable change is an increased RMSF for helix 4 for the S85G mutation for the active state most likely due to the introduction of a glycine residue.Figure S6. (A) Overlay of 1H-15N HSQC spectra of NtrCr wild-type (red) and S85D (blue) at 25°C indicating that the structures and the active/inactive equilibrium are not altered by this mutation. Data were linearly extrapolated to determine the rates of unfolding in the absence of denaturant for each NtrCr mutant.d. S85D inactive (darkest blue) and active (brown) and S85G inactive (light blue) and active (gold).
N CPMG NMR relaxation dispersion data (Palmer et al.. Mutation identified to be involved in the pathway by TMD (Lei et al. 30 (plum). 9 (raspberry). this mutation does not alter the overall rate. Apparently. 91 (navy blue).. In the TMD trajectory (Lei et al. The dispersion curves are color coded as followed: residue 6 (ivory). a mutation that does not alter the macroscopically observed kinetics.Figure S8.. 2009) that is not rate-limiting for the activation process.
. 50 (mustard). We wanted to test the effect of an alanine mutation in this position on the overall rate of inactive/active interconversion. 29 (salmon). 2001) for G97A NtrCr
indicating that the rate of inactive/active interconversion is similar to the rate for the wild-type form. This mutation serves a negative control. although it is possible that the energy barrier of this step in the transition pathway is increased but it must be still lower than the highest energy barrier. 82 (magenta). 102 (royal blue). 78 (cyan). 35 (lavender). 11 (black). and 122 (dark brown). G97 samples phi and psi angles that are only in allowed regions in the Ramachandran plot for a glycine residue.
B BeF 3
R2 eff (s -1)
R2 eff (s -1)
20 16 12 8 0 200 400 600 800 1000
16 14 12 10
R2 eff (s -1)
R2 eff (s -1)
20 16 12 8 4
6 0 200 400 600 800 1000 0 200 400 600 800 1000
-1 -1 10 s 10 s → → Utrans ← Ucis ← Fcis -2 -1 -2
2 3 4 5 GdmHCl ([M])
→ Ucis → Fcis ←-1 ←-3
10 s-1 10 s-1 10
. Fluorescence Emission from 300-450 (nm)
360 356 352 348 344
2 0 -2 -4 -6 -8 0 1 2 3 4 5 GdmHCl ([M]) 6 7 8
-12 0 1 2 3 4
[GdmHCl].4 1.0 0.4
[GdmHCl].6 1.8 1.6 0.8 0.2 0 1 2 3 4
x 10 0
[GdmHCl].6 0.2 0.2 0 1 2 3 4
x 10 2.8 1. M
x 10 1.0 1.A
x 10 1.0 0.4 1. M
5.0 0 20 40 60 80 100 120
.Figure S5 A
14 10 6 2 0 20 40 60 80 100 120
M G (s-1 / M)
5 3 1 0 20
x 10 7.0
5 9.0 8.0 7.Figure S6
108 110 112
116 118 120 122 124 126 128 10.5 10.5 7.0 130
-2 WT S85D
ln(ku ).5 1H (ppm) 8.5
3. (s -1)
-6 2.5 GdmHCl (M)
ν CPMG (Hz)
11 69 70 72 78 82 87 88 89 91 100
residue WT D86N D86NA89T BeF3-
1.3 1.07±0.3 1.75±0.03±0.45±0.08±0.2 2.7 2.62±0.15 1.22±0.48±0.d.5
.3 1.64±0.33±0.m.995±0.45 2. All errors are one s.3 1.05
0.18±0.35±0.2 2. Missing chemical shift values are indicative of residues that are severely exchange broadened or overlapped.2 2.65±0.32±0.79±0.4 2.1 2.p.32±0.4 1.4 1.67±0.21±0.24±0.5
1.3 2.6 1.2 1.39±0.4 1.30±0.03
* Chemical shifts are reported for those residues in NtrC that had quantifiable chemical exchange when fit to the Carver-Richards equation.2 1.ωA| p.4 2.02
0.14±0.2 1.Table S1 – Chemical Shift Differences Between the Inactive (ωI) and Active (ωA) States Calculated From Relaxation Dispersion Data and the Corresponding Populations ________________________________________________________________
Δω|ωI .25 1.38±0.
8 ku (s-1) ΔΔG‡ of unfolding compared to wt a (cal/mol) ΔΔG compared to wt from CPMG data b (cal/mol) 0 700 ± 60 750 ± 70 -3240 ± 320
7.2 700 ± 400 1.72E-06 2.32E-05 ± D86N 49.
Table 3.9 ± 7.17E-06 a) ku are used in the Eyring equation for the calculation.22E-05 3.d.36E-08 ± Activated (C-PO4) -3300 ± 400 1.66E-06 ± 0 1. b) Data used here. Folding and unfolding rates and comparison of the free energy of activation of unfolding to the corresponding free energy change caused by constitutively activating mutations or phosphorylation
kf (s-1) WT 51.66E-06 ± 0 1. Unfolding rates and corresponding free energy of activation of unfolding for mutant forms that effect the transition landscape
ku (s-1) WT ΔΔG‡ of unfolding compared to wt a (cal/mol)
7.4 600 ± 300 3. see also methods All errors are one s.9 ± 12.90E-05 ± D86N/A89T 48.05E-06 ± S85D 0 ± 400 2.d.Table S2.17E-08 a) ku are used in the Eyring equation for the calculation.61E-06 8. All errors are one s. see Figure 2E.
. see also methods.61E-06 2.4 ± 8.
. Kruber. Kinetic characterization of the chemotactic protein from Escherichia coli. D. Munoz.. Scherer. M. Drewello. 455-461... C.. V. Karplus. Jager. 5858-5866.... Palmer. A. and Kern. Schutkowski. 449460. (2007). 149-165. 231-240. J Mol Biol. S. E. Lopez. Nuclear magnetic resonance methods for quantifying microsecond-to-millisecond motions in biological macromolecules. L.P. Methods Enzymol 423. M. Folding of ribonuclease A and ribonuclease T1. M.G. CheY. Biochemistry 33. 204-238. F. T. Segmented Transition Pathway of the Signaling Protein Nitrogen Regulatory Protein C. and Fischer. M. Side-chain effects on peptidyl-prolyl cis/trans isomerisation.. K. Neubert.K. G. and Fischer. U. (1994)..X. Kroenke. J Mol Biol 279. Methods Enzymol 339. and Schmid. Kiefhaber. and Loria. G... Gardino. (2001)... Kivenson.D. and Serrano. A. J. II.. Kinetic coupling between protein folding and prolyl isomerization. (1994).. D. Functional dynamics of response regulators using NMR relaxation techniques. Schutkowski.. Influence on proline-specific enzymes of a substrate containing the thioxoaminoacyl-prolyl peptide bond.M. Kinetic analysis of the inverse hydrophobic effect.. (1992). G. M. A.. (1998). and Kern.Supplemental References Gardino. 3rd. M. Velos.. J Mol Biol 224. J. Reimer. (2009). Lei. A. Eur J Biochem 221.