The p12 domain is unstructured in a murine leukemia virus p12-CA(N) Gag construct

Abstract

The Gag polyproteins of gammaretroviruses contain a conserved p12 domain between MA and CA that plays critical roles in virus assembly, reverse transcription and nuclear integration. Here we show using nuclear magnetic resonance, that p12 is unstructured in a Moloney murine leukemia virus (MMLV) Gag fragment that includes the N-terminal domain of CA (p12-CA(N)). Furthermore, no long range interactions were observed between the domains, as has been previously predicted. Flexibility appears to be a common feature of Gag "late" domains required for virus release during budding. Residues near the N-terminus of CA(N) that form a beta-hairpin in the mature CA protein are unfolded in p12-CA(N), consistent with proposals that hairpin formation helps trigger capsid assembly.

Figure 1. ¹H-¹⁵N correlation (HSQC) spectrum obtained for MMLV p12CA^N.

Assignments are shown for signals in less-crowded regions of the spectrum. Red peaks represent signals folded in the ¹⁵N dimension.

Figure 2. NMR chemical shift and relaxation data that identify regions of structure and mobility in p12CA^N.

(A) Amino acid sequence of p12CA^N (arrow denotes proteolytic cleavage site). Residues of CA^N that adopt α-helical conformations in the N-MLV CA^N crystal structure are denoted by colored rectangles. (B) NMR chemical shift indices for the backbone Cα atoms of p12CA^N. Positive values denote helical regions, negative values denote regions of β-structure, and stretches of residues with near-zero values denote random coil conformations. For comparison, α-helical segments observed in the N-MLV CA^N crystal structure are aligned at the top of the panel. (C) ¹⁵N{¹H} heteronuclear NOE (XNOE) data obtained for p12CA^N. Values near 1.0 reflect reduced molecular motion, and smaller or negative values reflect motion on a fast (ps-ns) timescale.