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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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The two bound GDP nucleotides in the G domain.(A) General view showing the relative positions of the two GDP molecules. The two GDPs are about 31Å apart. (B) The GDP•magnesium bound in the active site is represented with its hydrated magnesium ion. (C) The external GDP (yellow) is bound near the insertion box domain. Another stacked external GDP (GDP*) molecule belonging to a symmetry-related molecule is also represented (light pink) to emphasize the crystal packing contact. In (B) and (C) the residues involved in the interaction with a nucleotide are labeled. Hydrogen bonds are shown.
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pone-0003619-g002: The two bound GDP nucleotides in the G domain.(A) General view showing the relative positions of the two GDP molecules. The two GDPs are about 31Å apart. (B) The GDP•magnesium bound in the active site is represented with its hydrated magnesium ion. (C) The external GDP (yellow) is bound near the insertion box domain. Another stacked external GDP (GDP*) molecule belonging to a symmetry-related molecule is also represented (light pink) to emphasize the crystal packing contact. In (B) and (C) the residues involved in the interaction with a nucleotide are labeled. Hydrogen bonds are shown.

Mentions: We tried to co-crystallize Pfu-FtsY in presence of GTP. Although SRP-GTPases, especially the SR subgroup, are distinguished by their low intrinsic GTPase activity and nucleotide specificity [13], the crystal structure we obtained showed the presence of GDP•magnesium bound in the catalytic site suggesting that nucleotide hydrolysis took place during the course of crystallization. Identical crystals could be obtained in presence of GDP but not in presence of non-hydrolyzable GTP analogs. The resulting structure was solved at 2.0Å resolution by molecular replacement using the apo structure as template. Two bound GDP molecules were identified (Figure 2A) and placed in the initial experimental electron density maps. Refinement to consistent atomic displacement factors shows that the GDP observed in the cognate binding site is present at full occupancy while the external GDP is present at only 69% occupancy despite the fairly high concentration (10 mM) of nucleotide used for crystallization; this lower occupancy probably reflects the lower affinity of this binding site.


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)

The two bound GDP nucleotides in the G domain.(A) General view showing the relative positions of the two GDP molecules. The two GDPs are about 31Å apart. (B) The GDP•magnesium bound in the active site is represented with its hydrated magnesium ion. (C) The external GDP (yellow) is bound near the insertion box domain. Another stacked external GDP (GDP*) molecule belonging to a symmetry-related molecule is also represented (light pink) to emphasize the crystal packing contact. In (B) and (C) the residues involved in the interaction with a nucleotide are labeled. Hydrogen bonds are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2572998&req=5

pone-0003619-g002: The two bound GDP nucleotides in the G domain.(A) General view showing the relative positions of the two GDP molecules. The two GDPs are about 31Å apart. (B) The GDP•magnesium bound in the active site is represented with its hydrated magnesium ion. (C) The external GDP (yellow) is bound near the insertion box domain. Another stacked external GDP (GDP*) molecule belonging to a symmetry-related molecule is also represented (light pink) to emphasize the crystal packing contact. In (B) and (C) the residues involved in the interaction with a nucleotide are labeled. Hydrogen bonds are shown.
Mentions: We tried to co-crystallize Pfu-FtsY in presence of GTP. Although SRP-GTPases, especially the SR subgroup, are distinguished by their low intrinsic GTPase activity and nucleotide specificity [13], the crystal structure we obtained showed the presence of GDP•magnesium bound in the catalytic site suggesting that nucleotide hydrolysis took place during the course of crystallization. Identical crystals could be obtained in presence of GDP but not in presence of non-hydrolyzable GTP analogs. The resulting structure was solved at 2.0Å resolution by molecular replacement using the apo structure as template. Two bound GDP molecules were identified (Figure 2A) and placed in the initial experimental electron density maps. Refinement to consistent atomic displacement factors shows that the GDP observed in the cognate binding site is present at full occupancy while the external GDP is present at only 69% occupancy despite the fairly high concentration (10 mM) of nucleotide used for crystallization; this lower occupancy probably reflects the lower affinity of this binding site.

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