Conformational dynamics of the HIV Vif protein complex

Abstract

HIV-1 viral infectivity factor (Vif) is an intrinsically disordered protein responsible for the ubiquitination of the APOBEC3 antiviral proteins. Vif folds when it binds the Cullin-RING E3 ligase CRL5 and the transcription cofactor CBF-β. A five-protein complex containing the substrate receptor (Vif, CBF-β, Elongin-B, Elongin-C) and Cullin5 (CUL5) has a published crystal structure, but dynamics of this VCBC-CUL5 complex have not been characterized. Here, we use Molecular Dynamics (MD) simulations and NMR to characterize the dynamics of the VCBC complex with and without CUL5 and APOBEC3 bound. Our simulations show that the VCBC complex undergoes global dynamics involving twisting and clamshell opening of the complex, while VCBC-CUL5 maintains a more static conformation, similar to the crystal structure. This observation from MD is supported by methyl-transverse relaxation optimized spectroscopy (methyl-TROSY) NMR data, which indicates that the entire VCBC complex without CUL5 is dynamic on the s-ms timescale. Vif binds APOBEC3 to recruit it to the complex, and methyl-TROSY NMR shows that the VCBC complex is more conformationally restricted when bound to APOBEC3F, consistent with our MD simulations. Vif contains a flexible linker region located at the hinge of the VCBC complex, which changes conformation in conjuction with the global dynamics of the complex. Like other ubiquitin substrate receptors,

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

“…As shown previously (14,23), VCBC forms a stable complex with ssDNA. This property of VCBC allows us to apply the hybrid DNA approach we used for the unambiguous characterization of the dynamics of ssDNA binding proteins using this HS-AFM methodology (22,24,25).…”

“…A similar plot for free VCBC and the corresponding AFM frame is presented in Figure 4B, D. As plots in Figure 4A and B show, there is a large fluctuation between angle of the arms in the triangle structure, which may reach up to 70 degrees for both free VCBC and VCBC in the VCBC-DNA complex. Note that the triangle structure observed in our study for VCBC in the VCBC-DNA complex and free VCBC are reminiscent the U-shape structure resolved from X-ray crystallography of the VCBC-Cul5 complex (13) and the clamshell state for VCBC complex observed in MD simulations studies (14). In general, the fluctuations of the angle we observed between the arms of the triangle structure correlates with the clamshell closing and opening, as is shown in (14), and supports the significant dynamics of this structure.…”

“…After formation of the complex between VCBC and hybrid DNA ( Figure S1 (1)) the VCBC appears on the AFM image as a dsDNA fragment with the protein sitting at the end ( Figure S1 (2)). Among the four-protein complex, only Vif is capable of binding ssDNA part of hybrid DNA as it was shown in (14,23). Figure 1A presents a cartoon for VCBC bound to ssDNA where EloB, EloC and CBF-β are indicated by grey colors, and Vif, which is bound to ssDNA (bright blue line), is marked as red.…”

“…The crystal structure of the VCBC-Cul5 complex has been solved (13). It has been shown that Vif, in the VCBC complex, has a large (α/β) domain and small (α) domain connected by a flexible linker (13,14). The linker part of Vif is important for the binding of Cul5 and for folding and creating a substrate for the E3 ubiquitin ligase complex (15).…”

“…In the first reports of the dynamics of VCBC-Cul1 and Cul5 complexes, using molecular dynamics (MD) simulations (16)(17)(18), the authors found a set of conformations that were different from the crystal structure that was previously reported (13), and showed that the Vif-ubiquitination complex is flexible. Recently (14), using MD simulations, investigators characterized and compared the dynamics of VCBC and VCBC-Cul5 complexes. They demonstrated the global dynamics of the VCBC complex but found the VCBC-Cul5 complex less flexible.…”