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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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The relaxation parameters of the amide 15N nuclei of each residue of the C-domain, measured at 14 T (600 MHz proton resonance frequency) and 298 K. (A) The longitudinal relaxation rate, R1 (s−1). (B) The transverse relaxation rate, R2 (s−1). (C) The heteronuclear 15N,1H-steady-state NOE values. (D) The order parameter S2, determined by model-free analysis. (E) Chemical exchange Rex contributions to the transverse relaxation rates (s−1).
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fig05: The relaxation parameters of the amide 15N nuclei of each residue of the C-domain, measured at 14 T (600 MHz proton resonance frequency) and 298 K. (A) The longitudinal relaxation rate, R1 (s−1). (B) The transverse relaxation rate, R2 (s−1). (C) The heteronuclear 15N,1H-steady-state NOE values. (D) The order parameter S2, determined by model-free analysis. (E) Chemical exchange Rex contributions to the transverse relaxation rates (s−1).

Mentions: Experimentally determined 15N-relaxation parameters for the amide 15N nuclei (R1, longitudinal relaxation rate; R2, transverse relaxation rate; and 15N{1H}-NOE values) measured at 298 K are shown in Fig. 5A–C. Figure 5D also shows the calculated values of the order parameter S2, which reflects the amplitude of picosecond–nanosecond amide bond vector dynamics, and Fig. 5E shows additional line broadening (Rex) resulting from protein motions on the millisecond time scale. The best fitting of the relaxation parameters could only be obtained using a fully asymmetric tensor model for the molecular rotational diffusion motions.


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)

The relaxation parameters of the amide 15N nuclei of each residue of the C-domain, measured at 14 T (600 MHz proton resonance frequency) and 298 K. (A) The longitudinal relaxation rate, R1 (s−1). (B) The transverse relaxation rate, R2 (s−1). (C) The heteronuclear 15N,1H-steady-state NOE values. (D) The order parameter S2, determined by model-free analysis. (E) Chemical exchange Rex contributions to the transverse relaxation rates (s−1).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig05: The relaxation parameters of the amide 15N nuclei of each residue of the C-domain, measured at 14 T (600 MHz proton resonance frequency) and 298 K. (A) The longitudinal relaxation rate, R1 (s−1). (B) The transverse relaxation rate, R2 (s−1). (C) The heteronuclear 15N,1H-steady-state NOE values. (D) The order parameter S2, determined by model-free analysis. (E) Chemical exchange Rex contributions to the transverse relaxation rates (s−1).
Mentions: Experimentally determined 15N-relaxation parameters for the amide 15N nuclei (R1, longitudinal relaxation rate; R2, transverse relaxation rate; and 15N{1H}-NOE values) measured at 298 K are shown in Fig. 5A–C. Figure 5D also shows the calculated values of the order parameter S2, which reflects the amplitude of picosecond–nanosecond amide bond vector dynamics, and Fig. 5E shows additional line broadening (Rex) resulting from protein motions on the millisecond time scale. The best fitting of the relaxation parameters could only be obtained using a fully asymmetric tensor model for the molecular rotational diffusion motions.

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