Nanostructures of APOBEC3G Support a Hierarchical Assembly Model of High Molecular Mass Ribonucleoprotein Particles from Dimeric Subunits

Abstract

Human APOBEC3G (hA3G) is a cytidine deaminase that restricts human immunodeficiency virus (HIV)-1 infection in a vif(the virion infectivity factor from HIV)-dependent manner. hA3G from HIV-permissive activated CD4؉ T-cells exists as an inactive, high molecular mass (HMM) complex that can be transformed in vitro into an active, low molecular mass (LMM) variant comparable with that of HIV-non-permissive CD4؉ T-cells. Here we present low resolution structures of hA3G in HMM and LMM forms determined by small angle x-ray scattering and advanced shape reconstruction methods. The results show that LMM particles have an extended shape, dissimilar to known cytidine deaminases, featuring novel tail-to-tail dimerization. Shape analysis of LMM and HMM structures revealed how symmetric association of dimers could lead to minimal HMM variants. These observations imply that the disruption of cellular HMM particles may require regulation of protein-RNA, as well as protein-protein interactions, which has implications for therapeutic development.

APOBEC3G Subunits Self-associate via the C-terminal Deaminase Domain

“…In the current study, we demonstrate through in vitro and in-cell studies that hA3G forms multimers through protein-protein contacts within the ZDD domain of the C terminus. Consistent with our previous SAXS model, domains within the N terminus of hA3G did not form protein-protein multimers (35). The proposed elongated conformation of hA3G was further supported by the finding that domains retained their unique attributes of known functions and interactions when expressed as individual monomeric domains.…”

“…1A), it was posited that hA3G subunits dimerize via the C-terminal domain (1,35). Gel filtration of RNase-digested, recombinant full-length hA3G purified from baculovirus-infected Sf9 cells also supported a dimeric mass with an elution time corresponding to 95 kDa (35). Such a mass was consistent with a dimeric peak in other chromatography studies (21,42,43).…”

“…Based on this distribution of ZDD domains within the SAXS envelope as well as the location of each ZDD motif in a single polypeptide (Fig. 1A), it was posited that hA3G subunits dimerize via the C-terminal domain (1,35). Gel filtration of RNase-digested, recombinant full-length hA3G purified from baculovirus-infected Sf9 cells also supported a dimeric mass with an elution time corresponding to 95 kDa (35).…”

“…The size and mass suggested a dimer comprising four discrete, relatively large volumes arranged in an elongated and linear manner (Fig. 1B) (35). Each large volume of the molecular envelope accommodates the approximate volume of a representative ZDD domain (Fig.…”

“…Small angle x-ray scattering (SAXS) and advanced envelope restoration methods revealed the shape of full-length and catalytically active hA3G in solution (35). The resulting molecular envelope suggested that the native conformation of hA3G is minimally a dimer with subunit contacts through the C termini.…”