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Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein.

Sharma A, Bruns K, Röder R, Henklein P, Votteler J, Wray V, Schubert U - BMC Struct. Biol. (2009)

Bottom Line: The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101).The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Structural Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. alok.sharma@gmail.com

ABSTRACT

Background: The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.

Results: In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this alpha-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.

Conclusions: These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

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Summary of the observed NOEs for the C-terminus of sp9. (A) Summary of the observed short and medium range NOEs for sp922-51 in 50% TFE at 300 K. (B) Root mean square deviations (rmsd) for the backbone atoms of sp922-51 in each residue calculated using the consecutive segment method plotted against the residue number for the 20 final structures. (C) Supposition of the 20 best final restrained structures of sp922-51 after alignment of the backbone atoms of residues Ser-27 to Ser-48. Shown are structures comprising residues Leu-22 to Glu-51.
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Figure 6: Summary of the observed NOEs for the C-terminus of sp9. (A) Summary of the observed short and medium range NOEs for sp922-51 in 50% TFE at 300 K. (B) Root mean square deviations (rmsd) for the backbone atoms of sp922-51 in each residue calculated using the consecutive segment method plotted against the residue number for the 20 final structures. (C) Supposition of the 20 best final restrained structures of sp922-51 after alignment of the backbone atoms of residues Ser-27 to Ser-48. Shown are structures comprising residues Leu-22 to Glu-51.

Mentions: Hence after quantification of the NOE data a total of 346 NOEs (Fig. 6A and Table 2) were used as distance restraints to calculate 100 structures using a standard protocol [24]. The 20 structures with the lowest NOE and total energies and without distance violations greater than 0.2 Å were chosen for the final fitting analysis (Table 2). The heterogeneity within these structures was assessed using the consecutive segment approach, in which the rmsd (root mean square deviations) of the backbone atoms for short segments, 2-5 residues in length, were systematically and pair wise determined [25]. This analysis allows identification of regions of high similarity within the 20 final conformations and therefore identification of stable structural elements. The best defined regions of the molecule were then those showing rmsd of the backbone atoms of less than 0.2 Å, namely a continuous stretch comprising amino acid residues Asp-25 to Glu-51 in which the 20 refined structures share a high degree of similarity (Fig. 6B). This finding is in good agreement with the 1Hα chemical shift data, the observed qualitative NOEs, and the Bradley-analysis which all suggest the presence of a well defined α-helix in the region Ser-27 to Leu-49.


Solution structure of the equine infectious anemia virus p9 protein: a rationalization of its different ALIX binding requirements compared to the analogous HIV-p6 protein.

Sharma A, Bruns K, Röder R, Henklein P, Votteler J, Wray V, Schubert U - BMC Struct. Biol. (2009)

Summary of the observed NOEs for the C-terminus of sp9. (A) Summary of the observed short and medium range NOEs for sp922-51 in 50% TFE at 300 K. (B) Root mean square deviations (rmsd) for the backbone atoms of sp922-51 in each residue calculated using the consecutive segment method plotted against the residue number for the 20 final structures. (C) Supposition of the 20 best final restrained structures of sp922-51 after alignment of the backbone atoms of residues Ser-27 to Ser-48. Shown are structures comprising residues Leu-22 to Glu-51.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2803184&req=5

Figure 6: Summary of the observed NOEs for the C-terminus of sp9. (A) Summary of the observed short and medium range NOEs for sp922-51 in 50% TFE at 300 K. (B) Root mean square deviations (rmsd) for the backbone atoms of sp922-51 in each residue calculated using the consecutive segment method plotted against the residue number for the 20 final structures. (C) Supposition of the 20 best final restrained structures of sp922-51 after alignment of the backbone atoms of residues Ser-27 to Ser-48. Shown are structures comprising residues Leu-22 to Glu-51.
Mentions: Hence after quantification of the NOE data a total of 346 NOEs (Fig. 6A and Table 2) were used as distance restraints to calculate 100 structures using a standard protocol [24]. The 20 structures with the lowest NOE and total energies and without distance violations greater than 0.2 Å were chosen for the final fitting analysis (Table 2). The heterogeneity within these structures was assessed using the consecutive segment approach, in which the rmsd (root mean square deviations) of the backbone atoms for short segments, 2-5 residues in length, were systematically and pair wise determined [25]. This analysis allows identification of regions of high similarity within the 20 final conformations and therefore identification of stable structural elements. The best defined regions of the molecule were then those showing rmsd of the backbone atoms of less than 0.2 Å, namely a continuous stretch comprising amino acid residues Asp-25 to Glu-51 in which the 20 refined structures share a high degree of similarity (Fig. 6B). This finding is in good agreement with the 1Hα chemical shift data, the observed qualitative NOEs, and the Bradley-analysis which all suggest the presence of a well defined α-helix in the region Ser-27 to Leu-49.

Bottom Line: The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101).The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Structural Biology, Helmholtz Centre for Infection Research, D-38124 Braunschweig, Germany. alok.sharma@gmail.com

ABSTRACT

Background: The equine infection anemia virus (EIAV) p9 Gag protein contains the late (L-) domain required for efficient virus release of nascent virions from the cell membrane of infected cell.

Results: In the present study the p9 protein and N- and C-terminal fragments (residues 1-21 and 22-51, respectively) were chemically synthesized and used for structural analyses. Circular dichroism and 1H-NMR spectroscopy provide the first molecular insight into the secondary structure and folding of this 51-amino acid protein under different solution conditions. Qualitative 1H-chemical shift and NOE data indicate that in a pure aqueous environment p9 favors an unstructured state. In its most structured state under hydrophobic conditions, p9 adopts a stable helical structure within the C-terminus. Quantitative NOE data further revealed that this alpha-helix extends from Ser-27 to Ser-48, while the N-terminal residues remain unstructured. The structural elements identified for p9 differ substantially from that of the functional homologous HIV-1 p6 protein.

Conclusions: These structural differences are discussed in the context of the different types of L-domains regulating distinct cellular pathways in virus budding. EIAV p9 mediates virus release by recruiting the ALG2-interacting protein X (ALIX) via the YPDL-motif to the site of virus budding, the counterpart of the YPXnL-motif found in p6. However, p6 contains an additional PTAP L-domain that promotes HIV-1 release by binding to the tumor susceptibility gene 101 (Tsg101). The notion that structures found in p9 differ form that of p6 further support the idea that different mechanisms regulate binding of ALIX to primary versus secondary L-domains types.

Show MeSH
Related in: MedlinePlus