High-speed atomic force microscopy directly visualizes conformational dynamics of the HIV Vif protein in complex with three host proteins

Vif (viral infectivity factor) is a protein that is essential for the replication of the HIV-1 virus. The key function of Vif is to dis-rupt the antiviral activity of host APOBEC3 (apolipoprotein B mRNA-editing enzyme catalytic subunit 3) proteins, which mutate viral nucleic acids. Inside the cell, Vif binds to the host cell proteins Elongin-C, Elongin-B, and core-binding factor subunit b , forming a four-protein complex called VCBC. The structure of VCBC – Cullin5 has recently been solved by X-ray crystallography, and, using molecular dynamics simulations, the dynamics of VCBC have been characterized. Here, we applied time-lapse high-speed atomic force microscopy to visualize the conformational changes of the VCBC complex. We determined the three most favorable conformations of this complex, which we identified as the triangle, dumbbell, and globular structures. Moreover, we characterized the dynamics of each of these structures. Our data revealed the very dynamic behavior of all of them, with the triangle and dumbbell structures being the most dynamic. These findings provide insight into the structure and dynamics of the VCBC complex and may support efforts to improve HIV treatment, because Vif is essential for virus survival in the cell.

HIV is an enveloped retrovirus that causes AIDS in humans (1). The virus particle contains two identical RNA copies and structural and replication enzymes for virus reproduction (2). One of the critical proteins needed for the virus survival is Vif (viral infectivity factor) (3). Vif, a small and unstructured 23-kDa protein (4,5), counteracts the potent HIV-1 inhibitors, human A3G and A3F proteins (APOBEC3 proteins) (6)(7)(8). Without Vif present, APOBEC3 proteins interact with the HIV virion and inhibit viral replication by deamination, turning cytidines to uridines in the viral DNA (9). Vif forms a complex with the APOBEC3 proteins, which serves as a substrate for binding with Cul5-E3 ubiquitin ligase to polyubiquitinate and degrade APOBEC3 proteins (10). To fold and form a stable structure, Vif interacts with different proteins: EloB (Elongin-B), EloC (Elongin-C) at its C terminus, and core-binding factor subunitb (CBFb) at its N terminus (11)(12)(13), forming the VCBC complex. Another protein, Cul5, interacts with Vif to form the VCBC-Cul5 complex and provides more stability and structure for the complex (13). The crystal structure of the VCBC-Cul5 complex has been solved (13). It has been shown that Vif, in the VCBC complex, has large (a/b) domain and small (a) domains 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. The authors also revealed the efficient binding of VCBC with single-stranded DNA (ssDNA) and found out that ssDNA stimulates the formation of VCBC dimers. However, direct experimental evidence to the VCBC complex dynamics are missing.
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 samples different conformational states with the most favorable ones, including globular, dumbbell, and triangle structures. Based on the data obtained for VCBC in complex with DNA, we identified free VCBC in the same experiments, just dissociated from the VCBC-DNA complex, and followed its dynamics. We revealed that free VCBC samples conformational states and undergo transitions between globular, dumbbell, and triangle structures. The frameby-frame analysis of time-lapse HS-AFM experiments allowed us to characterize these structures and compare dynamics for VCBC in VCBC-DNA complexes with free VCBC. The significance of the revealed dynamics of the VCBC complex is discussed.

Results
Conformational states of VCBC in complex with DNA 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 This article contains supporting information. * For correspondence: Yuri L. Lyubchenko, ylyubchenko@unmc.edu.
HS-AFM methodology (22,24,25). Fig. S1 shows a schematic presentation of the complex formation between VCBC and hybrid DNA. After formation of the complex between VCBC and hybrid DNA (Fig. S1, (1)), the VCBC appears on the AFM image as a dsDNA fragment with the protein sitting at the end (Fig. S1, (2)). Among the four-protein complex, only Vif is capable of binding ssDNA part of hybrid DNA as it was shown in Refs. 14 and 23. Fig. 1A presents a cartoon for VCBC bound to ssDNA where EloB, EloC, and CBF-b are indicated by gray shading, and Vif, which is bound to ssDNA (bright blue line), is marked as red shading. The dark blue line illustrates the dsDNA part of the hybrid DNA construct.
To visualize the dynamics of VCBC in the VCBC-DNA complex, we applied time-lapse HS-AFM imaging. The imaging was done in the VCBC binding buffer without drying of the sample (see details of sample preparation under "Experimental procedures"). After a complex of interest was found on the AFM image during scanning, we followed frame-by-frame (800 ms/frame) conformational changes in this complex and then assembled frames into movies. From all recorded movies for VCBC in VCBC-DNA complexes, we observed several conformational states. Three conformational states of VCBC were identified as the most favorable. These states were described as follows; a compact, globular conformation, when all four VCBC proteins are close to each other; an extended, dumbbell conformation, with two clearly separated blobs; and a triangle conformation with three clear protein blobs. The transitions between all these VCBC structures are clearly seen in all collected movies (see Supporting Information). Fig. 1B presents selected frames from Movie S1, which shows different VCBC structures and transitions between them. Specifically, frame 1 shows the globular structure of VCBC, with four proteins close to each other as schematically shown in the cartoon above the frame. The transformation of the VCBC complex from a globular structure into three visible blobs, denoted as a triangle structure, can be seen in frame 5. Note that proteins in the triangle structure undergo some rearrangement, showing different blob sizes and distances between them, as observed when comparing frames 5, 8, and 10. Next, in frames 7-10, the complex transitions back and forth from the triangle structure to the dumbbell structure. The complex reforms the globular structure in frame 23, which remains until the complex dissociates in frame 56. The appropriate cartoons above these frames illustrate possible arrangements and rearrangements of proteins in the VCBC complex. Another interesting example of the structural transitions that VCBC undergoes is seen in selected frames in Fig. 2. Again, three different structures, globular, triangle, and dumbbell, are seen from this movie (Movie S2). Frame 11 illustrates a globular structure, which transforms into a dumbbell in frame 15 and then into a triangle in frame 22 and stays as a triangle in frame 23, showing different distances between three protein blobs. Frames 23, 24, 26, and 27 show the transformation of VCBC triangle structure with three protein blobs (frame 23) into four protein blobs (frames 24 and 26) and reassembly of four proteins back into the triangle structure in frame 27. The observation of such assembly and reassembly of VCBC proteins confirms, without a doubt, that globular, dumbbell, and triangle structures include all four proteins. Thus, the recorded movies confirm that the VCBC in complex with DNA is a fully assembled complex containing all four proteins.

Conformational states of free VCBC
To follow the dynamics of free VCBC, we used the AFM images for the VCBC in complex with DNA and selected a VCBC complex that was dissociated from the VCBC-DNA complex as one particle and followed its dynamics. Analysis of recorded movies shows that free VCBC is also highly dynamic and, similar to VCBC in the VCBC-DNA complex, undergoes multiple conformations, including globular, dumbbell, and triangle. Fig. 3 shows one of the examples of the four-protein VCBC complex, which adopted a triangle structure as seen in a Figure 1. The dynamics of VCBC in the complex with DNA. A, cartoon of the VCBC complex with Vif bound to ssDNA, part of hybrid DNA (bright and dark blue indicate the ssDNA and dsDNA parts of hybrid DNA, respectively). B, selected frames for VCBC-DNA complex from Movie S1. Frame 1 presents the complex in the globular structure, which transforms into the triangle structure in frame 5 and then changes its structure between the dumbbell (frames 7 and 9) and triangle structures (frames 8 and 10), forming the globular structure in frame 23, as schematically illustrated above and below the AFM frames. Frame 56 shows dissociation of the VCBC from the VCBC-DNA complex forming a dumbbell structure (blue arrow). The protein below, marked by a red circle, is a newly assembled VCBC from nearby proteins as it is seen from Movie S1. The scale bar is 50 nm. The scan rate is 800 ms/frame. Small cartoons above and below selected frames demonstrate the dynamics of the triangle structure of free VCBC. The rearrangements of the proteins in the VCBC complex and the changes in the distances between protein blobs in the triangle structure are seen from the presented frames. The additional selected frames from the movies are shown in Figs. S2 and S3. In Fig. S2, the dissociation of VCBC from DNA occurs between frames 1 and 5. Free VCBC has a triangle structure in frames 5 and 14 and then adopts a dumbbell structure in frame 38 before going back to a triangle structure at the very end of the movie (Movie S4). Fig. S3 illustrates the selected frames for free VCBC from Movie S5. Frames 7 and 10 show a globular structure of VCBC, which transforms into dumbbell in frame 29 and looks like rotated dumbbell structure in frame 42.
To quantitatively characterize the dynamics of all structures, we measured the angle between arms in the triangle structure shown in Fig. 4. Fig. 4A presents the results for VCBC in complex with DNA, and Fig. 4B shows the results for free VCBC. C and D of Fig. 4 present corresponding AFM images explaining how the measurements are made. Similar analysis has been done for the dumbbell structures of VCBC in complex with DNA ( Fig. 5A) and free VCBC (Fig. 5B).
The globular structure of VCBC in complex with DNA and free VCBC was characterized by size, which is measured in AFM as a volume (26). The results are shown in Fig. 6A, which corresponds to the volume distribution for VCBC in complex with DNA. Fig. 6B shows similar measurements for free VCBC.
The occurrences of the different conformations of VCBC in complex with DNA and free VCBC are illustrated graphically in Fig. 7. The entire graph was made over 12 movies for VCBC in complex with DNA and 15 movies for free VCBC. Each movie contained dozens of frames.

Comparison of structures of VCBC in VCBC-DNA complex and free VCBC
Our time-lapse HS-AFM imaging showed highly dynamic conformational changes of free VCBC and VCBC in complex    with DNA. From all recorded movies, we observed different conformational states of free VCBC and VCBC in the VCBC-DNA complex and arranged them into the three most populated structures: the triangle, dumbbell, and globular structures. Frame-by-frame analysis of dozens of movies of free VCBC and VCBC in VCBC-DNA complexes allowed us to identify some characteristics of these structures.
To estimate the occurrence of the compact, extended, and triangle conformational states, we calculated the yield of each structure, and the data are presented in Fig. 7. For VCBC in VCBC-DNA complexes, the yields of all three structures show an even distribution. For free VCBC, the yields of globular and dumbbell structures are similar; however, the yield of the triangle structure is higher. This indicates that for free VCBC, the triangle structure is slightly more populated when compared with the others. Comparison of the yields of all three structures between free VCBC and VCBC in complex with DNA shows similar yields for globular and dumbbell structures. However, the yield of triangle structure is slightly lower for VCBC in the complex with DNA compared with free VCBC, which indicates that DNA may affect the assembly of the triangle structure of VCBC.
In addition to the yields of different structures, we analyzed the time-dependent probability of different VCBC structures. Based on selected 50 frames from Movie S1, we assembled the graph shown in Fig. S7. This graph illustrates the transitions between the globular (red circle), dumbbell (black square), and triangle (green triangle) structures depending on the frame number. As seen from this graph, there is no specific order for the transition from one structure to another, suggesting that there is no correlation for transitions from one particular structure to another. This conclusion was supported by analyses of the other movies described in Fig. S7.
In addition to the yield of each structure, we analyzed their dynamics. For the triangle structure, we followed the dynamics of VCBC in complex with DNA and compared it with the dynamics of free VCBC. The plot in Fig. 4A demonstrates the changes of the angle between two arms of the triangle structure for VCBC in the VCBC-DNA complex. White lines and the arrow in the AFM frame (Fig. 4C) indicate the measured angle. Similar plots for free VCBC and the corresponding AFM frame are presented in Fig. 4 (B and D). As the plots in Fig. 4 (A and B) show, there is a large fluctuation between angle of the arms in the triangle structure, which may reach up to 70°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 of the U-shaped structure resolved from X-ray crystallography of the VCBC-Cul5 complex (13) and the clamshell state for VCBC complex observed in MD simulation studies (14). In general, the fluctuations of the angle we observed between the arms of the triangle structure correlate with the clamshell closing and opening, as is shown in Ref. 14, and supports the significant dynamics of this structure. Another presentation demonstrating the dynamics of the triangle structure is shown in Fig. S4 for VCBC in the VCBC-DNA complex (Fig. S4A) and free VCBC (Fig. S4B). Fig. S4C is the histogram of the measured angle collected from 12 movies for VCBC in the VCBC-DNA complex and fitted with a Gaussian curve. The histogram for free VCBC, collected from 15 movies, fitted with a Gaussian curve is presented in Fig. S4D. The maxima in these histograms after Gaussian fitting are similar for both free VCBC and VCBC in complex with DNA; however, the histogram for VCBC in the VCBC-DNA complex is wider than for free VCBC, which translates into a more dynamic structure for VCBC in complex with DNA. These results suggest that DNA does not restrict the dynamics of VCBC in complex with DNA. In addition to opening and closing of the triangle structure, the authors of Ref. 14 provided the distances between proteins in the VCBC complex. Their measurements show that the distance between EloB 80 and CBF-b 37 is ;7 nm and the distance between EloB 58 and CBF-b 37 is between 2 and 5 nm for the VCBC complex. HS-AFM imaging does not allow for the identification of individual proteins in the VCBC complex. However, we can measure the distances between visible blobs in the VCBC complex when they are clearly separated from each other. One of the examples of such measurements is presented in Fig. S6. The distances measured for the triangle structure of VCBC in the VCBC-DNA complex (Fig. S6A) and free VCBC (Fig. S6B) are in the range of 8-10 nm, which is larger than observed from MD simulations results (14). This suggests that HS-AFM data revealed intermolecular dynamics within the VCBC complex rather than projections of a stable complex. Note that the distances between protein blobs in free VCBC and in the VCBC-DNA complex are close to each other, which emphasizes that DNA does not restrict the dynamics of the four proteins in the VCBC complex.
To follow the dynamics of the dumbbell structure, we measured the distance between the two clearly seen blobs, and the results are shown in Fig. 5A for VCBC in the VCBC-DNA complex and in Fig. 5B for free VCBC. The blue lines on the AFM frames illustrate the measurements of the distance for VCBC in the VCBC-DNA complex (Fig. 5C) and free VCBC (Fig. 5D), respectively. Rather large dynamics for both free VCBC and VCBC in the VCBC-DNA complex can be seen for the dumbbell structure, as shown in Fig. 5 (A and B). In this structure, two protein blobs of different sizes reflect a rather complex arrangement: the four proteins of VCBC are compacted into only two visible blobs. For this structure, the distance between two protein blobs shows fluctuation in the range of 4-14 nm, which demonstrates even larger dynamics of dumbbell structure than the triangle structure. It is an interesting observation, which may indicate that the dynamics of VCBC depend on the assembled structure. Another presentation demonstrating the dynamics of the dumbbell structure is shown in Fig. S5 for VCBC in the VCBC-DNA complex assembled from 12 movies (Fig. S5A) and free VCBC assembled from 15 movies (Fig. S5B). Indeed, Fig. S5 demonstrates AFM frames for VCBC in the VCBC-DNA complex (Fig. S5A), free VCBC (Fig. S5B), and assembled histograms (Fig. S5, C and D) from the collected movies. The comparison of these histograms clearly shows the difference in the dynamics of VCBC: the range of distances is wider for VCBC in complex with DNA compared with free VCBC. These data suggest that in the case of the dumbbell structure, DNA not only restricts but even slightly facilitates the dynamics of VCBC in the VCBC-DNA complex.
We also followed the dynamics of the globular VCBC structure for both free VCBC and VCBC in the VCBC-DNA complex. The results of the estimated volumes for this structure are presented in Fig. 6. The data are assembled into histograms, and the standard deviations, calculated after fitting with Gaussian curves, demonstrated the dynamic behavior of this structure. However, the most interesting fact is that VCBC in the VCBC-DNA complex has two separate peaks in the histogram (Fig. 6A) compared with only one peak for free VCBC (Fig. 6B). The value of the maximum for the volume in the case of free VCBC (318 6 80 nm 3 ) is close to the first maximum in the histogram for VCBC in the VCBC-ssDNA complex (298 6 100 nm 3 ). Based on this fact, we assigned the first peak in Fig. 6A and the peak in Fig. 6B to the monomeric state of VCBC. The second peak in histogram (Fig. 6A) is roughly double the value of the first peak (703 6 80 nm 3 ), which points to the possible formation of the VCBC dimers in the presence of DNA. The formation of VCBC dimers in the presence of DNA correlates with the results reported previously (14), where authors observed the formation of VCBC dimers in the presence of 14 oligonucleotides (dT14). According to the standard deviation values, the dynamics of VCBC monomers in the VCBC-DNA complex and free VCBC are close to each other, suggesting that DNA does not affect the dynamics of the globular structure.
Together, the comparison of the results between free VCBC and VCBC in the VCBC-DNA complex demonstrates that DNA does not restrict the dynamics of the triangle, dumbbell, and globular structures. In general, the dynamics of VCBC in the presence of DNA is similar or slightly larger than the dynamics of free VCBC. Based on crystallographic and MD simulation data, Vif adopts a folded conformation, interacting with EloB, EloC, and CBF-b in the four-protein VCBC complex. The flexible linker located between the a/b and a domain of Vif is responsible for conformational changes of VCBC (14). Based on the fact that DNA does not affect the dynamics of VCBC and the type of VCBC conformations, we suggest that DNA binds a region away from the flexible linker of Vif.
In summary, our studies directly demonstrate for the first time the very large conformational dynamics of the VCBC complex. Among numerous intermediate conformational changes of the VCBC complex, we revealed the most populated structures, which we identified as the triangle, dumbbell, and globular structures. Although our results show large conformational dynamics in all three favorable structures, we find that the most dynamic structures are the triangle and dumbbell structures, which suggests that the range of dynamics may depend on the conformational state of the VCBC complex. Also, our data not only confirmed the global dynamics of VCBC, characterized from MD simulations, but also demonstrated even larger conformational mobility of VCBC, whether for free VCBC or in the VCBC-DNA complex. Our results provide evidence for the structure-dynamics properties of the VCBC complex. The VCBC is a complex assembly with multiple functionalities involving interaction with other proteins. For example, changes in the structure and dynamics of VCBC can be linked with the complex ability for ubiquitination of A3 proteins or the packing of A3 proteins into the virions. Also, the different conformational states of VCBC identified in our experiments may provide the strategy for the design of small molecules to inhibit A3 ubiquitination and degradation and improve inhibition of viral replication by increasing A3 encapsidation. To identify the structure-function relationship for VCBC, more studies are needed, which are our long-range goals.

VCBC protein
The Hxb2 VCBC used in this study was obtained from Dr. J. Gross (University of California, San Francisco) and purified as described in Ref. 27.

Hybrid DNA substrate
Hybrid DNA was assembled as described in Ref. 28 and consists of a 69-nt ssDNA tail with a 379-bp dsDNA as a tag (see cartoon in Fig. S1, (1)). Briefly, 89-nt-long synthetic oligonucleotides were annealed with short 23-nt oligonucleotides (Integrated DNA Technology) to create a short duplex with sticky ends. This short duplex was ligated with a 356-bp dsDNA fragment that was complementary to the duplex sticky ends. The hybrid DNA was gel-purified (Qiagen kit) and resuspended in 10 mM Tris, pH 7.5, and 1 mM EDTA.
The preparation of VCBC-DNA sample Hybrid DNA (60 nM) was mixed with Hxb2 VCBC (240 nM) at ratio of 1:4 in binding buffer, containing 50 mM HEPES, pH 7.5, 100 mM NaCl, 5 mM MgCl 2 , and 1 mM DTT. After the incubation of the mixture at 25°C for 15 min, the sample was diluted up to 2 nM with the binding buffer and immediately deposited on a functionalized mica surface (29,30).

HS-AFM imaging
The detailed description of time-lapse HS-AFM imaging is described in Refs. 21, 22, and 31. In brief, 2 ml of diluted VCBC-DNA complex were deposited for 2 min on a mica surface and washed with binding buffer. The HS-AFM scanning was initiated immediately after the washing step without drying the sample. The scan size was 300 3 300 nm. After the complex of interest was selected, frame-by-frame recording was started with a scan rate of 800 ms/frame. The probes for imaging were grown under an electron beam using short cantilevers (BL-AC10DS-A2; Olympus, Tokyo, Japan) with a spring constant 0.1-0.2 N/m 21 and a resonance frequency of 400-1000 kHz.

Analysis of the HS-AFM data
The movies were assembled after frame-by frame recording of VCBC in the VCBC-DNA complex and free VCBC. Analysis of the observed structures was performed by measuring the yield of different structures and their structural characteristics. For globular structures, the volume of the VCBC in the VCBC-DNA complex and free VCBC were measured as described in Ref. 26, using the cross-section feature from Femtoscan online software (Advance Technologies Center, Moscow, Russia). For the 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. On average, for each structure ;10 movies were analyzed, and the data were assembled into histograms and fitted with Gaussian curves.

Data availability
All data are contained within the article.