pi is 0092867409014342
TRANSCRIPT
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Cell, Volume 139
Supplemental Data
Transient Non-native HydrogenBonds 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 S1:15 N 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 BeF 3-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
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populations as described in Experimental Procedures. The CPMG data at 800 MHz are
consistent with the inactive/active conformational exchange occurring in the very fast
NMR time regime (k ex>> Δω), in which R ex is proportional to Δω2. Error in relaxation
rates are from the larger of the difference in duplicate points or 2% of the signal to noise.
Figure S2. Kinetic and thermodynamic data for folding of inactive and active wild-
type and mutant forms of NtrCr
measured by fluorescence.
(A) Chevron plot depicting the natural log of the observed rate (k obs), where k obs is equal
to the sum of the unfolding rate and folding rate (k obs= k u + k f ) for WT (red▲
), D86N
(orange ■), D86N/A89T (yellow ♦), and CPO4-activated NtrCr
(black ●). Rates were
measured using stopped-flow fluorescence at various guanidinium-hydrochloride
concentrations at 25°C. Error in unfolding rates are s.d. from the mean. (B)
Thermodynamic unfolding curve for wild-type NtrCr
measured by fluorescence after
equilibration for 1 hour at 25°C at various GdmCl concentrations. Error in fluorescence
unfolding data are s.d. from the mean. (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, D86N, and D86N/A89T mutants
do not. Rates for proline isomerization at 25°C were estimated from previously
published data (Reimer et al., 1998; Schutkowski et al., 1994). According to (B) for
wild-type NtrCr , k obs should curve below 3.3 M GdmCl due to the increasing contribution
of k f with an inflection point at 2.5M (k u = k f ). 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. For wild-type and mutants
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forms, 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). In contrast, for P-
NtrCr
the cis/trans isomerization is one order of magnitude faster than the refolding rate
consequently is no longer rate limiting. This results in the typical curving of the Chevron
plot for the latter. This model is buttressed by our stopped flow refolding experiments.
When refolding was initiated by dilution of the denaturant after incubation, a double
exponential process as detected with a fast rate followed by a slow rate of about 10-2
s-1
,
the time regime indicative for prolyl peptide bond isomerization. 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, 1992). Folding rates plotted
with open symbols in (A) are from the double jump experiments. We note that a very
similar model was used in folding studies of the homologous protein CheY (Munoz et al.,
1994). Rates of folding and unfolding determined from the chevron plot are listed in
Table S2.
Figure S3. Quantitative fits of the folding/unfolding equilibrium measured by
NMR.
(A) Change in peak intensity for W7 (peak of the folded state in a15 N
1H HSQC) in wild-
type NtrCr
with increasing denaturant concentration. (B) Effect of increasing
concentrations of salt (NaCl) on the peak intensity of W7 follows a single exponential.
(C) Change in intensity with increasing GdmHCl (same data as in a) fit to a modified
sigmoidal characterized by a two-state transition centered ~2.4M GdmHCl,
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corresponding to the unfolding transition, and an exponential to account for the change in
intensity due to an increase in ionic strength of the solution. (D) Plot of peak intensity
after subtraction of the ionic strength effect which can now be fitted to a standard two-
state sigmoidal. Error from the r.m.s.d. 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.
Figure S4. Residues that were fit as described in Fig. S3 to determine their change in
free energy of unfolding (Δ
GUF) are plotted onto the inactive state structure in red.
Grey residues are unassigned, prolines, or overlapped. Residues in yellow are assigned
peaks whose intensities were small due to exchange broadening. 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, and thus no information on the unfolding/folding
transition.
Figure S5. The change in free energy of unfolding (ΔGUF, A) and m-value (MG, B)
for each residue in NtrCr
fitted as described in Fig. S3.
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. Error derived from the r.m.s.d. 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.
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Figure S6. S85D mutation that removes a non-native hydrogen bond of the
transition does not affect the ground states (inactive and active state).
(A) Overlay of 1H-
15 N HSQC spectra of NtrC
r wild-type (red) and S85D (blue) at 25°C
indicating that the structures and the active/inactive equilibrium are not altered by this
mutation. (B) The unfolding rates (see also Fig. S 2) of wild-type (red ) and S85D
(blue ) are identical within experimental error. Rates were measured using stopped-
flow fluorescence at various guanidinium-hydrochloride concentrations at 25°C. Data
were linearly extrapolated to determine the rates of unfolding in the absence of
denaturant for each NtrCr
mutant, which are listed in Table S3. Error in unfolding rates
are s.d. from the mean.
Figure S7. Unbiased MD simulations in explicit water for wild-type, S85D and
S85G support the experimental finding that the ground states are not significantly
changed by these amino acid substitutions.
(A) Top panel: Time traces of backbone root mean square deviation (RMSD) along the
MD trajectories of the inactive (blue) and active (red) states. (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), S85D
inactive (darkest blue) and active (brown) and S85G inactive (light blue) and active
(gold). 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.
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Figure S8. Mutation identified to be involved in the pathway by TMD (Lei et al.,
2009) that is not rate-limiting for the activation process.
15 N CPMG NMR relaxation dispersion data (Palmer et al., 2001) for G97A NtrC
r
indicating that the rate of inactive/active interconversion is similar to the rate for the
wild-type form. The dispersion curves are color coded as followed: residue 6 (ivory), 9
(raspberry), 11 (black), 29 (salmon), 30 (plum), 35 (lavender), 50 (mustard), 78 (cyan),
82 (magenta), 91 (navy blue), 102 (royal blue), and 122 (dark brown).
In the TMD
trajectory (Lei et al., 2009), G97 samples phi and psi angles that are only in allowed
regions in the Ramachandran plot for a glycine residue. We wanted to test the effect of
an alanine mutation in this position on the overall rate of inactive/active interconversion.
Apparently, this mutation does not alter the overall rate, 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. This mutation serves a negative control, a mutation that
does not alter the macroscopically observed kinetics.
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24
4
8
12
16
20
0 800600400200 1000
S85D
24
20
8
12
16
0 200 400 600 800 1000
WT
18
10
12
14
16
8006004002000 1000
BeF
18
0
6
10
14
1000800600400200
Y101F
3-
R
e f f ( s
)
2
- 1
R
e f f ( s
)
2
- 1
R
e f f ( s
)
2
- 1
R
e f f ( s
)
2
- 1
CPMG (Hz) CPMG (Hz)
CPMG (Hz) CPMG (Hz)
A
C D
B
Figure S1
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-8
-6
-4
-2
0
2
0 1 2 3 4 5 6 7 8
344
348
352
356
360
0 1 2 3 4 5 6
GdmHCl ([M])
GdmHCl ([M])
Utrans Ucis Fcis
Utrans Ucis Fcis
Utrans Ucis Fcis
Utrans Ucis Fcis
W e i g h t e d A v g . F l u o r e s c e n c e
E m i s s i o n f r o m 3 0 0 - 4 5 0 ( n m )
l n ( k ) , (
s ) - 1
A
B C
10-2
10-3
10
-110
-2
10-1
10-2
10-3
10-2
←→ →
→→
←
← ←
WT, Mut
P-NtrC
s-1
s-1
s-1
s-1
s-1 s-1
s-1s-1
Figure S2
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P e a k
I n t e n s i t y
P e a k
I n t e n s i t y
P
e a k
I n t e n s i t y
P e a k
I n t e n s i t y
[GdmHCl], M
[NaCl], M
[GdmHCl], M
[GdmHCl], M
0.2
43210
1.8
1.4
1.0
0.6
x 105
0.4
3210
2.0
1.6
1.2
0.8
x 105
1.0
43210
1.8
1.4
0.6
0.2
x 105
-12
43210
0
-4
-8
x 104
A
D
C
B
Figure S3
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Figure S4
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18
80 100 1206040200
14
10
6
2
Residue
Residue
Residue
7
100806040200
1
3
5
120
5.0
3.0
1.0
12010080604020
7.0
0
x 105
G
( k c a l / m o l )
M
( s
/ M )
I n t e n s i t y
U F
G
- 1
A
C
B
Figure S5
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10.5 10.0 9.5 9.0 8.5 8.0 7.5 7.0130
128
126
124
122
120
118
116
114
112
110
108
106
1 5 N ( p p m )
1H (ppm)
WT
S85D
-6
-4
-2
2.5 3 3.5 4
l n ( k
) ,
( s
)
u
- 1
A
B
GdmHCl (M)
WT
S85D
Figure S6
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A
B
Figure S7
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0 200 400 600 800 1000
8
10
12
14
16
18
R
e f f ( s
)
2
- 1
CPMG (Hz) ν
Figure S8
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Table S1 – Chemical Shift Differences Between the Inactive (ωI) and Active
(ωA) States Calculated From Relaxation Dispersion Data and theCorresponding Populations
________________________________________________________________
Δω|ωI - ωA| p.p.m.
________________________________________________________________
residue WT D86N D86NA89T BeF3-
pA 0.14±0.02 0.43±0.03 0.65±0.05 0.995±0.002
11 1.64±0.4 1.03±0.3 1.39±0.3 1.79±0.569 1.30±0.4 1.62±0.270 1.32±0.3 1.08±0.372 1.07±0.2 1.08±0.278 2.38±0.7 1.32±0.482 2.35±0.2 2.21±0.287 1.33±0.2 1.08±0.15 1.22±0.188 2.45±0.6 1.75±0.3 2.24±0.4 2.18±0.389 2.48±0.25 2.39±0.291 1.75±0.4 1.29±0.2
100 1.64±0.5 1.67±0.45 2.39±0.55
* Chemical shifts are reported for those residues in NtrC that hadquantifiable chemical exchange when fit to the Carver-Richards equation.Missing chemical shift values are indicative of residues that are severelyexchange broadened or overlapped. All errors are one s.d.
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Table S2. Folding and unfolding rates and comparison of the free energy ofactivation of unfolding to the corresponding free energy change caused by
constitutively activating mutations or phosphorylation
kf (s-1)
ku (s-1)
ΔΔG‡ of unfolding
compared to wt
(cal/mol)a
ΔΔG comparto wt from
CPMG data
(cal/mol)b
WT 51.9 ± 7.87.66E-06 ±1.61E-06
0 0
D86N 49.4 ± 8.42.32E-05 ±3.72E-06
600 ± 300 700 ± 60
D86N/A89T 48.9 ± 12.22.90E-05 ±
1.22E-05
700 ± 400 750 ± 70
Activated (C-PO4) -3.36E-08 ±1.17E-08
-3300 ± 400 -3240 ± 320
a) ku are used in the Eyring equation for the calculation, see also methods.b) Data used here, see Figure 2E.All errors are one s.d.
Table 3. Unfolding rates and correspondingfree energy of activation of unfolding for
mutant forms that effect the transition landscape
ku (s-1)
ΔΔG‡ of unfolding
compared to wt
(cal/mol) a
WT7.66E-06 ±1.61E-06
0
S85D8.05E-06 ±2.17E-06
0 ± 400
a) ku are used in the Eyring equation for the
calculation, see also methodsAll errors are one s.d.
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Supplemental References
Gardino, A.K., and Kern, D. (2007). Functional dynamics of response regulators using
NMR relaxation techniques. Methods Enzymol 423, 149-165.
Kiefhaber, T., and Schmid, F.X. (1992). Kinetic coupling between protein folding and
prolyl isomerization. II. Folding of ribonuclease A and ribonuclease T1. J Mol Biol 224,
231-240.
Lei, M., Velos, J., Gardino, A., Kivenson, A., Karplus, M., and Kern, D. (2009).
Segmented Transition Pathway of the Signaling Protein Nitrogen Regulatory Protein C. J
Mol Biol.
Munoz, V., Lopez, E.M., Jager, M., and Serrano, L. (1994). Kinetic characterization of
the chemotactic protein from Escherichia coli, CheY. Kinetic analysis of the inverse
hydrophobic effect. Biochemistry 33, 5858-5866.
Palmer, A.G., 3rd, Kroenke, C.D., and Loria, J.P. (2001). Nuclear magnetic resonance
methods for quantifying microsecond-to-millisecond motions in biological
macromolecules. Methods Enzymol 339, 204-238.
Reimer, U., Scherer, G., Drewello, M., Kruber, S., Schutkowski, M., and Fischer, G.
(1998). Side-chain effects on peptidyl-prolyl cis/trans isomerisation. J Mol Biol 279, 449-
460.
Schutkowski, M., Neubert, K., and Fischer, G. (1994). Influence on proline-specific
enzymes of a substrate containing the thioxoaminoacyl-prolyl peptide bond. Eur J
Biochem 221, 455-461.