APOBEC3G in complex with RNA: bridging high-speed AFM and simulation studies

Original Research Article

Abstract

A3G is a cytidine deaminase that effectively restricts HIV-1 infection by deaminating single-stranded DNA (ssDNA) during viral reverse transcription. Despite progress in understanding the structure-function relationship of A3G-RNA complexes, very little is known about the interaction pattern of wild-type A3G with RNA, and even less about the interaction dynamics. In our recent work, we combined molecular dynamics simulations with time-lapse high-speed atomic force microscopy (HS-AFM) to investigate the behavior of free, full-length A3G. These studies revealed dynamic transitions between two major conformations: globular (gA3G) and dumbbell-shaped (dA3G), with HS-AFM data validating the simulation results. Here, we apply a similar approach to characterize the interactions and dynamics of the full-length A3G protein in complex with RNA. The HS-AFM experiments revealed a highly dynamic behavior of A3G in A3G-RNA complexes. Namely, A3G changes its structure between gA3G and dA3G conformations in the complex, very similar to the conformational transitions observed for free A3G. In addition, these experiments demonstrated that A3G conformation in complex with RNA strongly depends on RNA length. Complementary simulations provide mechanistic insights into how these conformations affect RNA binding and complex dynamics. The results from these simulations, validated by HS-AFM data, revealed distinct interaction patterns and dynamics between RNA and the two A3G conformations. Moreover, data from computer simulations allowed us to identify the most probable A3G residues in contact with RNA of different lengths for gA3G and dA3G. These findings advance our understanding of A3G-RNA interactions and highlight the importance of A3G's conformational flexibility.