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NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor.

Mantsyzov AB, Ivanova EV, Birdsall B, Alkalaeva EZ, Kryuchkova PN, Kelly G, Frolova LY, Polshakov VI - FEBS J. (2010)

Bottom Line: The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure.Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor.The results provide new insights into the possible role of the C-domain in the process of translation termination.

View Article: PubMed Central - PubMed

Affiliation: Center for Magnetic Tomography and Spectroscopy, M. V. Lomonosov Moscow State University, Russia.

ABSTRACT
Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.

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Protein backbone chemical shift differences between the resonances from the two conformations of loop 357–367. Absolute values of chemical shift differences are shown for: (A) Hα resonances; (B) HN signals; and (C) amide 15N resonances.
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fig02: Protein backbone chemical shift differences between the resonances from the two conformations of loop 357–367. Absolute values of chemical shift differences are shown for: (A) Hα resonances; (B) HN signals; and (C) amide 15N resonances.

Mentions: For the great majority of the residues in the minidomain, the differences between the chemical shifts of the two conformational states are sufficiently large to allow sequential assignments based on the use of 1H,13C,15N triple-resonance experiments (3D experiment correlating the amide HN and the Cα signals, 3D experiment correlating the amide HN and the Cα signal of the preceding amino acid, 3D experiment correlating the amide NH with the Cα and Cβ signals, 3D experiment correlating the amide NH with the Cα and Cβ signals of the preceding amino acid, and 3D experiment correlating the amide NH with the C′ signal of the preceding amino acid). Figure 2 presents the distribution of the chemical shift differences between the two protein conformers for the backbone amide proton, nitrogen and Hα signals for the minidomain. These differences are concentrated in regions 333–344 and 357–370, presumably reflecting differences in the structures in these regions. It should be noted that there are no detectable differences in chemical shifts for the remaining residues.


NMR solution structure and function of the C-terminal domain of eukaryotic class 1 polypeptide chain release factor.

Mantsyzov AB, Ivanova EV, Birdsall B, Alkalaeva EZ, Kryuchkova PN, Kelly G, Frolova LY, Polshakov VI - FEBS J. (2010)

Protein backbone chemical shift differences between the resonances from the two conformations of loop 357–367. Absolute values of chemical shift differences are shown for: (A) Hα resonances; (B) HN signals; and (C) amide 15N resonances.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2984548&req=5

fig02: Protein backbone chemical shift differences between the resonances from the two conformations of loop 357–367. Absolute values of chemical shift differences are shown for: (A) Hα resonances; (B) HN signals; and (C) amide 15N resonances.
Mentions: For the great majority of the residues in the minidomain, the differences between the chemical shifts of the two conformational states are sufficiently large to allow sequential assignments based on the use of 1H,13C,15N triple-resonance experiments (3D experiment correlating the amide HN and the Cα signals, 3D experiment correlating the amide HN and the Cα signal of the preceding amino acid, 3D experiment correlating the amide NH with the Cα and Cβ signals, 3D experiment correlating the amide NH with the Cα and Cβ signals of the preceding amino acid, and 3D experiment correlating the amide NH with the C′ signal of the preceding amino acid). Figure 2 presents the distribution of the chemical shift differences between the two protein conformers for the backbone amide proton, nitrogen and Hα signals for the minidomain. These differences are concentrated in regions 333–344 and 357–370, presumably reflecting differences in the structures in these regions. It should be noted that there are no detectable differences in chemical shifts for the remaining residues.

Bottom Line: The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure.Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor.The results provide new insights into the possible role of the C-domain in the process of translation termination.

View Article: PubMed Central - PubMed

Affiliation: Center for Magnetic Tomography and Spectroscopy, M. V. Lomonosov Moscow State University, Russia.

ABSTRACT
Termination of translation in eukaryotes is triggered by two polypeptide chain release factors, eukaryotic class 1 polypeptide chain release factor (eRF1) and eukaryotic class 2 polypeptide chain release factor 3. eRF1 is a three-domain protein that interacts with eukaryotic class 2 polypeptide chain release factor 3 via its C-terminal domain (C-domain). The high-resolution NMR structure of the human C-domain (residues 277-437) has been determined in solution. The overall fold and the structure of the beta-strand core of the protein in solution are similar to those found in the crystal structure. The structure of the minidomain (residues 329-372), which was ill-defined in the crystal structure, has been determined in solution. The protein backbone dynamics, studied using (15)N-relaxation experiments, showed that the C-terminal tail 414-437 and the minidomain are the most flexible parts of the human C-domain. The minidomain exists in solution in two conformational states, slowly interconverting on the NMR timescale. Superposition of this NMR solution structure of the human C-domain onto the available crystal structure of full-length human eRF1 shows that the minidomain is close to the stop codon-recognizing N-terminal domain. Mutations in the tip of the minidomain were found to affect the stop codon specificity of the factor. The results provide new insights into the possible role of the C-domain in the process of translation termination.

Show MeSH