the fic protein doc uses an inverted substrate to phosphorylate … · 2013-11-14 · 1 the fic...

1

The Fic protein Doc uses an inverted substrate to phosphorylate and

inactivate EF-Tu

Daniel Castro-Roa1†, Abel Garcia-Pino2,3†*, Steven De Gieter2,3, Nico A.J. van Nuland2,3, Remy Loris2,3, Nikolay Zenkin1*.

1Centre for Bacterial Cell Biology, Institute for Cell and Molecular Biosciences, Newcastle University, Baddiley-Clark Building, Richardson Road, Newcastle upon Tyne, NE2 4AX, UK; 2Structural Biology Brussels, Department of Biotechnology (DBIT), Vrije Universiteit Brussel, Pleinlaan 2, B-1050 Brussels, Belgium; 3Molecular Recognition Unit, Department of Structural Biology, VIB, Pleinlaan 2, B-1050 Brussels, Belgium

† D.C-R and A.G-P contributed equally to this work and should be considered co-first

authors.

*Correspondence to: Nikolay Zenkin, PhD Centre for Bacterial Cell Biology Institute for Cell and Molecular Biosciences Newcastle University Baddiley-Clark Building Richardson Road Newcastle upon Tyne NE2 4AX, UK Phone: +44(0)1912083227 FAX: +44(0)1912083205 E-mail: [email protected] Abel Garcia-Pino, PhD Structural Biology Brussels Department of Biotechnology Vrije Universiteit Brussel Building E, Pleinlaan 2 Brussels B-1050, Belgium Phone: +32 (0)2 6291025 FAX: +32 (0)2 6291963 E-mail: [email protected]

Nature Chemical Biology: doi:10.1038/nchembio.1364

2

Supplementary Results.

Supplementary Figure 1. Images of full gels, TLCs and TLEs produced in this work. Note that some gels were cut at the bottom before phosphorimaging to reduce the signal of radiolabeled NTPs migrating at the bottom of the gel.


3

Supplementary Figure 2. Kinetics of EF-Tu phosphorylation in the presence of ATP or

GTP. Data are mean of three independent experiments and error bars are standard deviations.

Data were fitted into a single-exponential equation and normalized to the predicted

maximum, which was taken as 100. ± sign represents standard error of the fit.


4


5

Supplementary Figure 3. Interplay between EF-Tu, Doc and nucleotides: representative

ITC titrations. Titration of EF-Tu into Doc in 1mM GDP (a), EF-Tu (free state) into Doc

(b), and EF-Tu into Doc in 1 mM of GMPPNP (c). (d) EF-Tu binding to Doc monitored by

the changes in intensity ratio (I/Io) of the 1H/15N HSQC spectrum of Doc. Residues S27, R38,

R64, L77 as function of EF-Tu concentration were used as probe. (e) AMPPNP binding to

Doc followed by chemical shift perturbations (Δδ) as function of AMPPNP concentration of

the 1H/15N HSQC spectrum of Doc. Residues Y20, F68, N78 were used as probe. Titration of

non-hydrolysable nucleotides into the pre-formed Doc:EF-Tu:GDP complex AMPPNP (f),

GMPPNP (g), and UMPPNP (h). Titration of Doc mutants with EF-Tu in 1 mM GDP,

DocN78W (i), DocH66Y (j), DocR64G (k), and Doc with the EF-TuT382V mutant (l). Titration of

AMPPNP into the DocN78W:EF-Tu:GDP complex (m). Titration of Doc with EF-Tu in the

NMR conditions (n). Titrations in the presence of Phd52-73 (the antitoxin domain of Phd) and

1 mM GDP, EF-Tu into Doc (o) and AMPPNP into the preformed Doc:EF-Tu complex (p).

See Supplementary Table 1 and Online Methods for further details.


6


7

Supplementary Figure 4. LC-MS/MS analysis of peptides from EF-Tu and EF-Tu

treated with Doc and ATP. The analysis of the LC-MS/MS spectra (the EF-Tu spectra in (a)

and the spectra of the Doc-treated EF-Tu in (b)) shows that the peptide consisting of the

region 374FAIREGGRTVGAGVVAK390 has a mass of 1688.9674 Da (m/z ratio 844.4837) in

the non-treated EF-Tu, and a mass of 1768.9312 Da (m/z ratio 884.4656) in the Doc-treated

EF-Tu. The difference in mass between both peptides equals 79.9638 Da, which is almost

identical to the average increase in mass expected from the introduction of a phosphate group

(79.9799 Da). Bottom part of each panel is magnification of the upper part. Other clusters of

peaks are other peptides. Peaks in clusters are natural isotopes of the same peptide.


8

Supplementary Figure 5. Characterization of the EF-Tu and Doc mutants by CD

spectroscopy. (a) The Figure shows that the EF-TuT382V mutant has a nearly identical far UV

CD spectrum as the wild type protein (Figure inset, EF-TuT382V in red and EF-Tu in blue) and

both proteins unfold approximately at the same temperature (EF-TuT382V at 52.6°C and EF-

Tu at 53.2°C), which suggests that this surface mutation has a negligible effect on the overall

structure and stability of the protein. (b) The R64G (in blue) and H66Y (in red) surface

mutations do not affect the overall secondary structure of Doc (in black) as monitored by far

UV CD. All CD measurements were done on a Jasco 715 spectropolarimeter, in Tris-HCl pH

7.4, 40 mM NH4Cl, 10 mM MgCl2, 1 mM TCEP.


9

Supplementary Figure 6. Dephosphorylation of EF-Tu by Doc in the presence of GDP.

The scheme of the experiment is shown above the radiogram (see also Fig. 3). EF-Tu 32P-

phosphorylated by Doc for 30 min to ensure full usage of γ[32P]-ATP was then incubated

with or without 5 µM Phd and/or 1 mM GDP for 2 hours and products analyzed by TLC. For

GDP mobility standard α[32P]-GTP was used in the reaction of EF-Tu phosphorylation,

which resulted in formation of α[32P]-GDP. Nonradioactive standards, visualized under

UV254 are marked with radioactive spots before phosphorimaging. Not all EF-Tu can be

dephosphorylated even after prolonged incubation due to either aggregation or to competition

from phosphorylation. The identity of the of EF-Tu spot at the start of chromatogram is

verified by addition of Ni2+-NTA-agarose beads that sequester the His-tagged EF-Tu before

spotting on TLC plate


10


11

Supplementary Figure 7. Assignment of Doc and NMR chemical shift perturbations. (a)

1H-15N HSQC spectrum of Doc and cross peak assignment (b) Chemical shift perturbations

observed in the 1H-15N HSQC spectrum of Doc upon addition of 0 μM, 34.0 μM, 58.0 μM

123.3 μM, 197.3 μM of EF-Tu. (c) Chemical shift perturbations observed in the 1H-15N

HSQC spectrum of Doc upon addition of 0 mM, 1.4 mM, 2.7 mM 9.0 mM, 15.0 mM, 25.8

mM and 40 mM of AMPPNP. (d) Mapping on the surface of Doc of the observed chemical

shifts perturbations (in red) used for the docking of AMPPNP on Doc. Residues R19, Y20,

G22, L23, G25, F68, R74, N78, D99, T101 and V102 are shown in red (see Figure 5 and

Supplementary Table 3 for further details).


12

Supplementary Figure 8. Determination of experimental SAXS parameters. Guinier

analysis of the experimental SAXS curves (in red) and the theoretical curves (in black)

derived from the models, for Doc (a), EF-Tu:GDP (b) and Doc:EF-Tu:GDP (c). In every case

the curves corresponding to the experimental data are displayed up by one logarithmic unit

for clarity. (d) P(r) functions obtained from the scattering curves using GNOM21 for Doc (in

black), EF-Tu:GDP (in blue) and Doc:EF-Tu:GDP (in red). (e) Stereo view of Doc:EF-

Tu:GDP representative solutions that fit to the experimental data with χ2 between 0.9 and 1.1.

In the Figure Doc is represented as ribbons and EF-Tu as a blue surface. The solutions

superimpose with a core r.m.s.d below 1.5 Å over 510 Cα atoms. Plots of r.m.s.d. versus χ2


13

(f) and χ2 versus model number (g). Selected solutions were clustered into three groups (blue,

green and orange circles). Blue lines demark the χ2 range of the final solutions.


14

Supplementary Figure 9. Chemical shift based model of Doc bound to ATP. The ATP

bound to Doc in the complex is shown as purple sticks. The orientation of the nucleotide in

the active site is antiparallel to that observed in FIC-like proteins (shown in green, based on

the structure of NmFic in complex with AMPPNP, pdbid 3S6A1 ), presenting the γ-phosphate

moiety toward H66 and the site where EF-Tu binds. Doc is colored in light grey and active

site residues H66, K73 and R74 are shown as black lines. In typical Fic domains K73 is

replaced by a glycine, which removes the steric hindrance and allows nucleotide binding, and

constitutes a major difference in the active site motif between both subfamilies.


15

Supplementary Figure 10. Phd binding site overlaps the NTP binding site on Doc. When

bound to Doc, the C-terminal domain of Phd (in yellow, based on the coordinates of the

Doc:Phd complex, pdbid 3K3324) occupies the NTP site (represented by the bound ATP

molecule in purple). Note that the site where the NTP binds in Fic-like domains (in green)

remains free in the Doc-Phd complex.


16

Supplementary Table 1. Interplay between Doc, EF-Tu and nucleotides. The binding

affinities were determined from fitting a single interaction model to the experimental data

from ITC and NMR titrations. Data represent mean values ± s.d. See Supplementary Figure 3

for representative titrations.

Experiment Technique Kd Number of experiments

EF-Tu titrated into Doc ITC 8 ± 4 μM 3

EF-Tu titrated into Doc in phosphate ITC 6 ± 1 μM 3

EF-Tu titrated into Doc in phosphate NMR 16.3 μM 1

EF-Tu titrated into Doc in 1mM GDP ITC 1.7 ± 0.7 μM 3

EF-Tu titrated into Doc in 1mM GMPPNP ITC 50 ± 7 μM 3

EF-Tu titrated into DocH66Y in 1mM GDP ITC 4 ± 2 μM 3

EF-TuT382V titrated into Doc in 1mM GDP ITC 10 ± 7 μM 3

EF-Tu titrated into DocR64G in 1mM GDP ITC no binding 2

EF-Tu titrated into Doc in 1mM GDP in Phd52-73 ITC no binding 2

EF-Tu titrated into DocN78W in 1mM GDP ITC 3 ± 1 μM 3

AMPPNP titrated into Doc NMR 7.2 mM 1

AMPPNP titrated into (preformed Doc:EF-Tu:GDP) ITC 0.26 ± 0.05 μM 3

GMPPNP titrated into (preformed Doc:EF-Tu:GDP) ITC 4.4 ± 0.4 μM 3

UMPPNP titrated into (preformed Doc:EF-Tu:GDP) ITC no binding 2

AMPPNP titrated into (preformed DocN78W:EF-Tu:GDP) ITC 45 ± 1 μM 3

AMPPNP titrated into Doc:EF-Tu:GDP and Phd52-73 ITC no binding 2

Supplementary Table 2. SAXS parameters. Theoretical and experimental molecular

weights of Doc, EF-Tu, and the Doc:EF-Tu as obtained from the SAXS curves. Using an RSAS

cutoff of 0.005 and Chi-values of 1.5 or lower, model-data agreements can be reliably

identified (Rambo & Tainer, Nature 2013)

Specie

Experimental Molecular

Weight SAXS (kDa)

Experimental Molecular

Weight MALS (kDa)

Theoretical Molecular

Weight (kDa)

Rg (Å) (exps/model) Dmax(Å) χ2 RSAS


17

Doc 15.0 14.3 14.7 16.7/16.3 56.4 0.8 0.0027

EF-Tu 44.1 43.9 43.7 23.6/23.8 77.7 1.1 0.0021

Doc:EF-Tu:GDP 56.0 56.7 57.0 25.8/24.6 74.3 0.9 0.0029

Additional SAXS parameters:

Specie Vc(model

) Vc(exp) VSAS Rg(model) Rg(exp) Io(model) Io(exp)

Doc 166.86 174.93 0.00213 16.3 16.7 595.18 632.69

EF-Tu 390.0 373.2 0.00203 23.8 23.6 861.32 833.1

Doc:EF-Tu:GDP 409.7 421.4 0.00077 24.6 25.8 118.5121.84

Supplementary Table 3. Chemical shift perturbations used for docking. Residues with

chemical shift perturbations above 2σ selected for the docking experiments.

Residue Experiment

S27 Docking of EF-Tu to Doc

R64 Docking of EF-Tu to Doc

H66 Docking of EF-Tu to Doc

R19 Docking of AMPPNP to Doc

Y20 Docking of AMPPNP to Doc

G22 Docking of AMPPNP to Doc

L23 Docking of AMPPNP to Doc

G25 Docking of AMPPNP to Doc

F68 Docking of AMPPNP to Doc

R74 Docking of AMPPNP to Doc

N78 Docking of AMPPNP to Doc

D99 Docking of AMPPNP to Doc


18

T101 Docking of AMPPNP to Doc

V102 Docking of AMPPNP to Doc


the fic protein doc uses an inverted substrate to phosphorylate … · 2013-11-14 · 1 the fic...

Documents