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Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: implications for the targeting step at the membrane.

Egea PF, Tsuruta H, de Leon GP, Napetschnig J, Walter P, Stroud RM - PLoS ONE (2008)

Bottom Line: The basic charges on the surface of this helix are likely to regulate interactions at the membrane.Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution.Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. pascal@msg.ucsf.edu

ABSTRACT
In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP*magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes.

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Related in: MedlinePlus

AUC and SAXS characterization of the Pfu and Taq FtsYs.Pfu data are represented in red and Taq in blue. (A) Sedimentation velocity analysis. Data are presented in g(S*) format using the time-derivative method analysis. The curves fitted against Gaussian functions (solid lines) are shown with their corresponding residual deviations. (B) Sedimentation equilibrium analysis. Radial distributions of concentrations along with the fitted exponential curves (solid lines) are plotted with their corresponding residual deviations. Two representative fits are shown for each protein. In all cases curves were fitted assuming a single species system. (C) and (D) High-angle analysis of SAXS data. (C) Scattered intensity curve fits. The experimental I(Q) curves (circles) displayed with errors bars were fitted using CRYSOL against the theoretical curves (solid lines) calculated from the crystal structures. (D) Distance distribution analysis. The experimental distance distribution functions P(r) are displayed (circles) with errors bars and compared with the ones calculated (solid lines) from the crystal structures using CRYSOL and GNOM. (E) Ab Initio 3D shape restoration by simulated annealing minimization. The protein envelopes are described using spherical harmonics expansion (fifth order). The retrieved shapes are rendered with ASSA [52] (drawn as transparent envelopes) with their corresponding crystal structures after optimal superposition along their respective inertia axes using SUPCOMB [53]. The final χ2s are 0.943 and 1.109 for Pfu and Taq receptors respectively. Four different orientations are shown to emphasize the quality of the reconstructions. Red asterisks indicate the areas of poorest overlap that correspond to the disordered regions in the crystal structures.
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pone-0003619-g005: AUC and SAXS characterization of the Pfu and Taq FtsYs.Pfu data are represented in red and Taq in blue. (A) Sedimentation velocity analysis. Data are presented in g(S*) format using the time-derivative method analysis. The curves fitted against Gaussian functions (solid lines) are shown with their corresponding residual deviations. (B) Sedimentation equilibrium analysis. Radial distributions of concentrations along with the fitted exponential curves (solid lines) are plotted with their corresponding residual deviations. Two representative fits are shown for each protein. In all cases curves were fitted assuming a single species system. (C) and (D) High-angle analysis of SAXS data. (C) Scattered intensity curve fits. The experimental I(Q) curves (circles) displayed with errors bars were fitted using CRYSOL against the theoretical curves (solid lines) calculated from the crystal structures. (D) Distance distribution analysis. The experimental distance distribution functions P(r) are displayed (circles) with errors bars and compared with the ones calculated (solid lines) from the crystal structures using CRYSOL and GNOM. (E) Ab Initio 3D shape restoration by simulated annealing minimization. The protein envelopes are described using spherical harmonics expansion (fifth order). The retrieved shapes are rendered with ASSA [52] (drawn as transparent envelopes) with their corresponding crystal structures after optimal superposition along their respective inertia axes using SUPCOMB [53]. The final χ2s are 0.943 and 1.109 for Pfu and Taq receptors respectively. Four different orientations are shown to emphasize the quality of the reconstructions. Red asterisks indicate the areas of poorest overlap that correspond to the disordered regions in the crystal structures.

Mentions: The apparent sedimentation coefficients of Pfu-FtsY (s = 3.7±0.1S) and Taq-FtsY (s = 2.4±0.1S) were determined (Table 2 and Figure 5A) and both receptors appeared as monomers in solution. The apparent monomeric association state established by velocity sedimentation was rigorously confirmed by equilibrium sedimentation experiments carried out over a wide, but still dilute, range of protein concentrations. Equilibrium experiments yielded molecular weight estimations of 34,900±1,780 Da and 32,640±1,610 Da for Pfu and Taq, respectively, in good agreement with the calculated values of 35,810 Da and 33,055 Da (Table 2 and Figure 5B).


Structures of the signal recognition particle receptor from the archaeon Pyrococcus furiosus: implications for the targeting step at the membrane.

Egea PF, Tsuruta H, de Leon GP, Napetschnig J, Walter P, Stroud RM - PLoS ONE (2008)

AUC and SAXS characterization of the Pfu and Taq FtsYs.Pfu data are represented in red and Taq in blue. (A) Sedimentation velocity analysis. Data are presented in g(S*) format using the time-derivative method analysis. The curves fitted against Gaussian functions (solid lines) are shown with their corresponding residual deviations. (B) Sedimentation equilibrium analysis. Radial distributions of concentrations along with the fitted exponential curves (solid lines) are plotted with their corresponding residual deviations. Two representative fits are shown for each protein. In all cases curves were fitted assuming a single species system. (C) and (D) High-angle analysis of SAXS data. (C) Scattered intensity curve fits. The experimental I(Q) curves (circles) displayed with errors bars were fitted using CRYSOL against the theoretical curves (solid lines) calculated from the crystal structures. (D) Distance distribution analysis. The experimental distance distribution functions P(r) are displayed (circles) with errors bars and compared with the ones calculated (solid lines) from the crystal structures using CRYSOL and GNOM. (E) Ab Initio 3D shape restoration by simulated annealing minimization. The protein envelopes are described using spherical harmonics expansion (fifth order). The retrieved shapes are rendered with ASSA [52] (drawn as transparent envelopes) with their corresponding crystal structures after optimal superposition along their respective inertia axes using SUPCOMB [53]. The final χ2s are 0.943 and 1.109 for Pfu and Taq receptors respectively. Four different orientations are shown to emphasize the quality of the reconstructions. Red asterisks indicate the areas of poorest overlap that correspond to the disordered regions in the crystal structures.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2572998&req=5

pone-0003619-g005: AUC and SAXS characterization of the Pfu and Taq FtsYs.Pfu data are represented in red and Taq in blue. (A) Sedimentation velocity analysis. Data are presented in g(S*) format using the time-derivative method analysis. The curves fitted against Gaussian functions (solid lines) are shown with their corresponding residual deviations. (B) Sedimentation equilibrium analysis. Radial distributions of concentrations along with the fitted exponential curves (solid lines) are plotted with their corresponding residual deviations. Two representative fits are shown for each protein. In all cases curves were fitted assuming a single species system. (C) and (D) High-angle analysis of SAXS data. (C) Scattered intensity curve fits. The experimental I(Q) curves (circles) displayed with errors bars were fitted using CRYSOL against the theoretical curves (solid lines) calculated from the crystal structures. (D) Distance distribution analysis. The experimental distance distribution functions P(r) are displayed (circles) with errors bars and compared with the ones calculated (solid lines) from the crystal structures using CRYSOL and GNOM. (E) Ab Initio 3D shape restoration by simulated annealing minimization. The protein envelopes are described using spherical harmonics expansion (fifth order). The retrieved shapes are rendered with ASSA [52] (drawn as transparent envelopes) with their corresponding crystal structures after optimal superposition along their respective inertia axes using SUPCOMB [53]. The final χ2s are 0.943 and 1.109 for Pfu and Taq receptors respectively. Four different orientations are shown to emphasize the quality of the reconstructions. Red asterisks indicate the areas of poorest overlap that correspond to the disordered regions in the crystal structures.
Mentions: The apparent sedimentation coefficients of Pfu-FtsY (s = 3.7±0.1S) and Taq-FtsY (s = 2.4±0.1S) were determined (Table 2 and Figure 5A) and both receptors appeared as monomers in solution. The apparent monomeric association state established by velocity sedimentation was rigorously confirmed by equilibrium sedimentation experiments carried out over a wide, but still dilute, range of protein concentrations. Equilibrium experiments yielded molecular weight estimations of 34,900±1,780 Da and 32,640±1,610 Da for Pfu and Taq, respectively, in good agreement with the calculated values of 35,810 Da and 33,055 Da (Table 2 and Figure 5B).

Bottom Line: The basic charges on the surface of this helix are likely to regulate interactions at the membrane.Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution.Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Biophysics, University of California San Francisco, San Francisco, California, USA. pascal@msg.ucsf.edu

ABSTRACT
In all organisms, a ribonucleoprotein called the signal recognition particle (SRP) and its receptor (SR) target nascent proteins from the ribosome to the translocon for secretion or membrane insertion. We present the first X-ray structures of an archeal FtsY, the receptor from the hyper-thermophile Pyrococcus furiosus (Pfu), in its free and GDP*magnesium-bound forms. The highly charged N-terminal domain of Pfu-FtsY is distinguished by a long N-terminal helix. The basic charges on the surface of this helix are likely to regulate interactions at the membrane. A peripheral GDP bound near a regulatory motif could indicate a site of interaction between the receptor and ribosomal or SRP RNAs. Small angle X-ray scattering and analytical ultracentrifugation indicate that the crystal structure of Pfu-FtsY correlates well with the average conformation in solution. Based on previous structures of two sub-complexes, we propose a model of the core of archeal and eukaryotic SRP*SR targeting complexes.

Show MeSH
Related in: MedlinePlus