Dynamics Studies of HIV-1 CA Protein Assemblies by Solid-State MAS NMR Spectroscopy

Abstract

The capsids of mature retroviruses perform the essential function of organizing the viral genome for efficient replication. In mature HIV-1 virions, the 25.6 kDa CA protein assemblies into a fullerene cone, a closed shell. Tyrosine residues in the capsid proteins of retroviruses play important roles in protein folding and assembly packing1. In HIV-1 CA protein, Y145, which is located in the linker region between N- and C-terminal domains might play a pivot role in HIV-1 capsid assembly.2,3 However, the molecular interactions involving Y145 that stabilize the assembly, including the dynamic behavior of this residue in the assembled and unassembled CA are unknown. We have recently determined that MAS NMR spectroscopy can yield atomic- resolution information on assembled CA capsids and reported partial resonance assignments and secondary structure analysis of conical assemblies.4 Herein, we present studies of the internal dynamics of the Tyr resiudes in CA as probed by various solid-state NMR methods. 15N/1H chemical shift anisotropies (CSA) and 1H-15N/1H-13C dipolar order parameters have been obtained in selectively labeled 13C, 15N -Tyr HIV-1 CA protein assemblies of conical morphologies, as well as in N- and C-terminal domains of CA. Our results demonstrate that Y145 exhibits reduced 15N and 1H chemical shift anisotropy, as well as smaller 1H-15N and 1H-13C dipolar order parameters, presumably due to its backbone mobility on nano- to microsecond timescales. Moreover, the NMR parameters of Y145 are strongly temperature dependent, another evidence of internal conformational dynamics. In contrast, the other three Tyr residues, Y130, Y164 and Y169, which are located in α-helical regions of CA, display rigid-limit chemical shielding anisotropies and dipolar order parameters.