Computational Model and Dynamics of Monomeric Full-Length APOBEC3G

Abstract

APOBEC3G (A3G) is a restriction factor that provides innate immunity against HIV-1 in the absence of viral infectivity factor (Vif) protein. However, structural information about A3G, which can aid in unraveling the mechanisms that govern its interactions and define its antiviral activity, remains unknown. Here, we built a computer model of a full-length A3G using docking approaches and molecular dynamics simulations, based on the available X-ray and NMR structural data for the two protein domains. The model revealed a large-scale dynamics of the A3G monomer, as the two A3G domains can assume compact forms or extended dumbbell type forms with domains visibly separated from each other. To validate the A3G model, we performed time-lapse high-speed atomic force microscopy (HS-AFM) experiments enabling us to get images of a fully hydrated A3G and to directly visualize its dynamics. HS-AFM confirmed that A3G exists in two forms, a globular form (∼84% of the time) and a dumbbell form (∼16% of the time), and can dynamically switch from one form to the other. The obtained HS-AFM results are in line with the computer modeling, which demonstrates a similar distribution between two forms. Furthermore, our simulations capture the complete process of A3G switching from the DNA-bound state to the closed state. The revealed dynamic nature of monomeric A3G could aid in target recognition including scanning for cytosine locations along the DNA strand and in interactions with viral RNA during packaging into HIV-1 particles.

Understanding the structural basis of HIV-1 restriction by the full length double-domain APOBEC3G

“…The structures reveal that CD1 and CD2 domains can form different interactions through a flexible linker to pack with each other at different angles. Two different CD1-CD2 packing orientations are observed in the two fl rA3G structures, suggesting the possibility for a certain degree of plasticity between the two domains, consistent with a prior computational simulation study 50 . M4 mutant carrying mutations on the CD1 interface with CD2 to alter the packing interactions still displayed full deaminase activity ( Fig.…”

High-Speed AFM directly visualizes conformational dynamics of the HIV-Vif protein complex

“…The detailed description of time-lapse HS-AFM imaging is described in (21,22,30). In brief, two microliters of diluted VCBC-DNA complex were deposited for 2 min on a mica surface and washed with binding buffer.…”

“…For globular structures, the volume of the VCBC in the VCBC-DNA complex and free VCBC were measured as described in (31), using the cross section feature from Femtoscan online software (Advance Technologies Center, Moscow, RU). For dumbbell structures, the same cross section feature was used to measure the distance between two clear blobs in the structure (21). For triangle structures the feature for angle measurement was used from Femtoscan software.…”

“…Here, we applied time-lapse high-speed atomic force microscopy (HS-AFM), which has been shown to be successful for studies of the dynamics of proteins and protein-DNA complexes (19)(20)(21)(22). Our data show that VCBC in complex with DNA is very dynamic and sampled different conformational states with the most favorable ones, including globular, dumbbell, and triangle structures.…”

Antiretroviral APOBEC3 Cytidine Deaminases Alter HIV-1 Provirus Integration Site Profiles

“…Within the non-catalytic N-terminal A3G domain, W94 is part of the SWSPCxxC zinc-coordinating motif while W127 is within ARLYYFW. These tryptophan residues are important for general nucleic acid binding ability, substrate sequence recognition and protein oligomerization 14,21,[42][43][44] . While both W94 and W127 mutations diminish RNA binding, the W127 substitution is unique because it prevents the homodimerization of A3G which results in a reduced processivity 14 .…”