Dynamic allostery governs cyclophilin A–HIV capsid interplay

Significance

The mechanisms of how Cyclophilin A (CypA) regulates HIV-1 infectivity remain poorly understood. We examined the role of dynamics in capsid (CA) protein assemblies by magic-angle-spinning NMR. The assembled CA is highly dynamic. Dipolar tensors calculated from molecular dynamics trajectories are in quantitative agreement with the NMR results. Motions in the CypA loop are sequence-dependent and attenuated in the escape mutants A92E and G94D. Dynamics are similar in escape mutants and CA/CypA complex. These findings suggest that CA escapes from CypA dependence through dynamic allostery. Thus, a host factor's function in HIV infectivity may not be primarily associated with a structural change of the capsid core, but with altering its dynamics, such as the reduction of motions for the CypA loop.

Abstract

Host factor protein Cyclophilin A (CypA) regulates HIV-1 viral infectivity through direct interactions with the viral capsid, by an unknown mechanism. CypA can either promote or inhibit viral infection, depending on host cell type and HIV-1 capsid (CA) protein sequence. We have examined the role of conformational dynamics on the nanosecond to millisecond timescale in HIV-1 CA assemblies in the escape from CypA dependence, by magic-angle spinning (MAS) NMR and molecular dynamics (MD). Through the analysis of backbone ¹H-¹⁵N and ¹H-¹³C dipolar tensors and peak intensities from 3D MAS NMR spectra of wild-type and the A92E and G94D CypA escape mutants, we demonstrate that assembled CA is dynamic, particularly in loop regions. The CypA loop in assembled wild-type CA from two strains exhibits unprecedented mobility on the nanosecond to microsecond timescales, and the experimental NMR dipolar order parameters are in quantitative agreement with those calculated from MD trajectories. Remarkably, the CypA loop dynamics of wild-type CA HXB2 assembly is significantly attenuated upon CypA binding, and the dynamics profiles of the A92E and G94D CypA escape mutants closely resemble that of wild-type CA assembly in complex with CypA. These results suggest that CypA loop dynamics is a determining factor in HIV-1's escape from CypA dependence.

Cyclophilin A (CypA), a peptidyl-prolyl isomerase, is a host factor critical in the regulation of the HIV-1 infection, involving a direct interaction with the capsid (CA) protein (1–3). The mechanism by which CypA modulates the viral infectivity is complex and poorly understood, being dependent on the CA protein primary sequence and the host cell type (4–6). For example, it is known that mutations in the CypA-binding loop of the CA protein dramatically reduce virus infectivity (7, 8). The A92E and G94D escape mutants bind CypA with similar affinity to wild-type CA, but exhibit only 10% of the activity of wild-type CA in the presence of CypA, and full infectivity can be restored if CypA is inhibited with cyclosporin A in the host cells (8), as shown schematically in SI Appendix, Fig. S1. Alas, the molecular mechanisms underlying CypA escape remain elusive, despite numerous virological, biochemical, and structural–biological studies.

The present study investigates the internal conformational dynamics of a CA protein assembly. Although static structures of HIV-1 proteins and complexes with host factors provide important clues into their assembly architecture and conformational details of the interactions, structures alone are insufficient for understanding molecular mechanisms. It is well known that biological functions can be dynamically regulated, at multiple levels of organization, from internal dynamics of individual protein molecules (9) to entire cells. This dynamic regulation certainly also applies to HIV-1 because numerous dynamic processes are associated with HIV-1 assembly, disassembly, release, and maturation (10, 11). For example, we previously demonstrated that internal conformational dynamics of the CA protein and its structural plasticity determine its ability to assemble into pleiomorphic conical capsids (12, 13) (Fig. 1). We also uncovered that, in the HIV-1 CA-SP1 maturation intermediate, dynamic disorder in the SP1 peptide plays an important role in the final step of virus maturation, permitting condensation of CA into the cores of infectious virions (14).

Fig. 1.

(A, Left) All-atom MD-derived model of mature HIV-1 capsid constructed on the basis of cryo-electron tomography (cryo-ET) and solution NMR studies (13). The capsid comprises 216 hexamers (orange) and 12 pentamers (blue) [Protein Data Bank (PDB) ID 3J3Y]. Structural organization of a hexamer of hexamers (HOH) building block is illustrated in the expansion. Color coded are individual hexameric units comprising the HOH building block. (A, Right) The 3D structure of CA monomer [HXB2 sequence polymorph [PDB file 3NTE (42)]. (B) Cosedimentation assay of CA with CypA illustrating the efficiency of cosedimentation for different CA/CypA molar ratios. S, supernatant; P, pellet. (C) Transmission electron microscopy (TEM) images of tubular assemblies of CA and CA/CypA. (C, Upper) CA NL4-3 (Left), CA NL4-3 A92E (Center), and CA NL4-3 G94D (Right). (C, Lower) HXB2 (Left) and CA HXB2/CypA (Right). (D) Expansions around the aliphatic region for 2D NCA and combined R2-driven (CORD) MAS NMR spectra for CA HXB2 (black) and CA HXB2/CypA (orange), illustrating the multiple chemical shift perturbations observed upon formation of the complex. These perturbations are mapped onto the structure of CA monomer (A) and are confined to flexible loops and residue variation sites. The spectra are recorded at 20.0 T and the MAS frequency of 14 kHz. (E) Expansions of glycine regions for 2D NCA MAS NMR spectra for (from left to right): HXB2, HXB2/CypA, NL4-3, NL4-3 A92E, and NL4-3 G94D. Dashed lines indicate the G89 cross-peaks associated with cis- and trans-P90.

In this study, we examined the residue-specific mobility of CA protein from HXB2 and NL4-3 sequence polymorphs (SI Appendix, Fig. S2) in tubular assemblies on the nanoseconds to milliseconds timescales. In particular, we compared wild-type and A92E and G94D escape mutants of the NL4-3 strain as well as wild-type HXB2 CA alone and in complex with CypA. As discussed previously (14, 15), tubular assemblies recapitulate the hexameric lattice, the predominant symmetry arrangement of the conical HIV-1 capsid core, illustrated in Fig. 1A. Dipolar tensors and resonance intensities extracted from a series of 2D and 3D homonuclear and heteronuclear magic-angle spinning (MAS) NMR experiments revealed that certain regions in both HXB2 and NL4-3 wild-type CA are unusually dynamic on all timescales. These motions are significantly attenuated upon CypA binding. Most remarkably, the dynamic profiles of the A92E and G94D escape mutants closely resemble that of CA when bound by CypA. To gain further understanding of the sequence-dependent dynamics profiles of CA assemblies, we performed extensive molecular dynamics (MD) simulations. The motionally averaged dipolar tensors extracted from the MD trajectories are in remarkable quantitative agreement with the NMR results. Together, our results suggest that changes in the sequence-dependent conformational dynamics may be a key determinant in the escape mechanism of HIV-1 CA capsid mutants from CypA dependence.

Results

CA/CypA Assemblies: CypA Binding and Sample Morphology.

In the presence of high concentration of NaCl (0.5–2.4 M), the CA protein assembles into tubes (14). As shown in Fig. 1 B and C, the assembly is highly efficient for wild-type CA, as well as the A92E and G94D escape mutants, enabling their MAS NMR characterization. To examine whether CypA binding interferes with the tubular assemblies, we conducted a cosedimentation experiment, in which varying concentrations of CypA were added to preassembled CA tubes. As shown in Fig. 1B, CypA coprecipitates with CA tubes, and, with increasing CypA concentrations, more CypA becomes part of the CA/CypA complex. Furthermore, our studies indicate that at the CA/CypA ratios below 2:1 CypA disrupts the tubes. Therefore, we used a 2:1 CA:CypA ratio for our MAS NMR studies. At this ratio, we observed efficient binding of CypA to tubular CA assemblies without any disruption of the tubes, as illustrated in Fig. 1C.

MAS NMR Spectra of CA and CA/CypA Assemblies.

The five CA assemblies under investigation (wild-type and A92E and G94D escape mutants of the NL4-3 strain, as well as wild-type HXB2 CA alone and in complex with CypA) yielded extremely well-resolved MAS NMR spectra, confirming previous observations (14). We speculate that the high spectral resolution in these assemblies is attained owing to their high conformational homogeneity and well-defined structure, similar to that observed in other supramolecular protein assemblies (16, 17), and making MAS NMR an attractive method for their structural and dynamics analysis with atomic resolution. Indeed, the quality of the data is such that differences in chemical shifts upon CypA binding and for CypA mutants are readily observed. As illustrated in Fig. 1D and SI Appendix, Fig. S3, assemblies of CA with bound CypA or the A92E and G94D escape mutants exhibit multiple chemical shift changes, respectively. These changes map onto the residues in the CypA loop, which are affected by the CypA binding or by the mutations in the loop residues. It should be noted that the four residues that differ in the primary sequence in the HXB2 and NL4-3 strains of HIV-1 exhibit signal changes as well (SI Appendix, Fig. S3), and so do their neighbors. It is important to note that the chemical shift differences are modest and do not exceed 2 ppm, indicating that the tertiary structure is not perturbed significantly. Indeed, secondary chemical shift analysis of the CypA loop residues reveals that the structure is similar in all five samples.

We also ascertained that the P90 residue in the CypA loop of CA does not undergo cis–trans isomerization to any detectable extent in the absence of CypA, as evidenced by a single N–C^α cross-peak for G89 associated with the trans-P90 in all samples investigated. This finding is in contrast to solution NMR results on the CA N-terminal domain, where the cis conformer for P90 was readily detected by us and others as a minor conformer (populated at ∼14% at room temperature) from two sets of resonances in G89 (SI Appendix, Fig. S7; refs. 18 and 19). The cis–trans isomerization is catalyzed by CypA and Nup358Cyp (18, 19). The uncatalyzed reaction is slow (exchange rates of <0.1 s⁻¹), whereas in the presence of CypA and NUP358Cyp, isomerization rates of 6.6 and 12.1 s⁻¹ were observed, respectively. In contrast, in the tubular assembly, P90 isomerization is absent. This finding is important because, as discussed below, the CypA loop undergoes motions on the nanoseconds to milliseconds timescales, and it is necessary to establish that these motions are not a result of cis–trans proline isomerization. Interestingly, in CA/CypA assemblies, the N–C^α cross-peak(s) of G89 is (are) missing (Fig. 1E), suggesting the presence of motions on the microseconds to milliseconds timescales that interfere with the polarization transfers in NCACX experiments. It is not likely that these motions may be associated with a catalyzed cis–trans proline exchange process because the timescale for the latter is too slow to interfere with the NCACX experiment.

Dynamics in CA/CypA Assemblies and CypA Escape Mutants on Nanosecond to Millisecond Timescales: ¹H-¹⁵N and ¹H-¹³C Dipolar Interaction Parameters and Spectral Intensities.

The one-bond ¹H-¹⁵N^H and ¹H-¹³C^α dipolar coupling constants in the absence of any motion are 11.34 (20) and 22.7 (21) kHz, respectively. If motions that are faster than the magnitude of the rigid-limit dipolar coupling constant (microseconds to nanoseconds) are present, the dipolar tensor is dynamically averaged, resulting in a reduced effective dipolar coupling (22). The dipolar coupling constants and the asymmetry parameters of the motionally averaged dipolar tensors are very sensitive probes of motional amplitudes and symmetries in the nanosecond to microsecond timescales, as we and others have demonstrated (22–24). We used 3D RN-symmetry based experiments (25) for the measurement of ¹H-¹⁵N^H and ¹H-¹³C^α dipolar lineshapes of individual resonances of CA assemblies. The dipolar interaction parameters were extracted by numerical simulations of the dipolar lineshapes (Materials and Methods).

The ¹H-¹⁵N and ¹H-¹³C dipolar order parameters (S) are plotted vs. residue number in Fig. 2 and SI Appendix, Fig. S4, respectively. The corresponding dipolar interaction parameters are compiled in SI Appendix, Table S1. As illustrated in Fig. 3 and SI Appendix, Figs. S5 and S6, the ¹H-¹⁵N and ¹H-¹³C dipolar lineshapes for the five samples are remarkably diverse, suggesting that a large number of residues undergo motions on the nanoseconds to microseconds timescales, as evidenced by their dynamically averaged lineshapes and greatly reduced dipolar order parameters. At the same time, for many resonances, the lineshapes and the corresponding dipolar interaction parameters are characteristic of rigid-limit behavior, with the majority of the rigid residues belonging to alpha helices, which are the main secondary structure element in the CA protein.

Fig. 2.

The ¹H-¹⁵N S in tubular assemblies of HXB2 CA (A) and NL4-3 A92E CA (B) plotted vs. the residue number. Experimental MAS NMR ¹H-¹⁵N S for assigned residues are shown as markers. Red markers for HXB2 associated with several residues represent a minor conformer. ¹H-¹⁵N S computed from MD trajectories are shown as lines. The computed S of NL4-3 A92E were extracted from the following MD trajectories: averaged over six CA molecules comprising different hexamers in one HOH building block (solid lines color-coded differently) and averaged over all molecules in a HOH building block (dashed yellow line). The MD trajectories for the HOH building block are extracted from all-atom MD-derived model of mature HIV-1 capsid (PDB ID 3J3Y) (13).

Fig. 3.

Summary of nanosecond to microsecond timescales dynamics for CypA loop residues observed in tubular assemblies of HIV-1 CA protein. Experimental ¹H-¹⁵N S plotted vs. the residue number (A) and corresponding dipolar lineshapes in B (from top to bottom): HXB2, HXB2/CypA, NL4-3, NL4-3 A92, and NL4-3 G94D. The experimental and simulated lineshapes are shown as solid black and dashed blue lines, respectively. The simulated dipolar lineshapes and the corresponding S for the minor R100 and S102 conformers are shown with dashed red lines and red markers, respectively. Note the large variations in S among the five CA assemblies and that CypA loop in the wild-type CA has increased mobility vis-à-vis the escape mutants and CA/CypA assemblies.

For wild-type CA from both the HXB2 and NL4-3 strains, mobile residues are found in the CypA-binding loop as well as in several short loops that connect helices 1 and 2; 2 and 3; 3 and 4; 9 and 10; and 10 and 11, as well as between the N-terminal beta-hairpin and helix 1. Our results are in agreement with prior solution NMR studies on unassembled N-terminal domain (NTD) and full-length CA NL4-3, which identified the same mobile and rigid regions of the protein on nanosecond to microsecond timescales, on the basis of R₁, R₂, and heteronuclear NOEs (26, 27). It had also been reported that loops in assembled CA exhibit a certain degree of mobility (28). However, completely unexpected was our finding that the dipolar order parameters are reduced tremendously for resonances of the CypA-binding loop residues, particularly those of G89 and A92. Resonances of these two residues, which are located in the middle of the loop, exhibit essentially isotropic lineshapes, indicating that the H–N bond vectors sample a very large conformational space. The fact that we are able to detect these loop residue resonances in the 3D spectra, despite their high flexibility, is very unusual. Indeed, it is fortuitous that the associated motions fall into a regime where they are fast enough not to interfere with cross-polarization (CP), decoupling, and MAS. Other resonances of residues in the CypA loop are also dynamic on the microsecond to nanosecond timescales, and their lineshapes are characteristic of rhombic dipolar tensors (e.g., H87 and A88). Interestingly, several residues that reside in the hinges of the different loops exhibit two conformations—a dynamic one (with reduced S) and a static one (with rigid-limit S)—despite a single chemical shift (Figs. 2 and 3). Overall, the above findings reflect an unprecedented degree of motion for the loops in wild-type CA protein assemblies from both HXB2 and NL4-3 strains.

In contrast, in the complex assemblies of CA/CypA, the motions of residues in all loop regions are significantly reduced compared with the CA assemblies in the absence of CypA (Figs. 2 and 3). The CypA loop residue A92 no longer exhibits isotropic lineshapes, and the dipolar order parameter is 0.44 (SI Appendix, Table S1). For G89, no amide resonances were detected, suggesting that motions occurring in the intermediate regime with respect to the CP and/or decoupling and/or MAS are present, rendering the corresponding peak in the 3D experiments invisible. This finding is not surprising per se, given that CypA binds directly to the CypA loop (2) and may attenuate motion. Alternatively, the G89 peak disappearance may be associated with the conformational exchange associated with cis–trans isomerization of the P90 peptide bond, but this possibility is not very likely because the isomerization timescale is too slow to interfere with the MAS NMR experiments. Also unexpected was that motions of other loop regions in the CA protein were attenuated (SI Appendix, Table S1).

Surprisingly, the dynamics profiles of assemblies of the two CypA escape mutants, A92E and G94D, resemble that of the CA/CypA rather than wild-type CA assemblies. The dipolar lineshapes associated with the CypA-binding loop residues in both mutants reveal that motions in this loop are reduced considerably, compared with wild-type CA assemblies, as illustrated in Fig. 3 and SI Appendix, Table S1. Remarkably, the dipolar order parameters of the CypA-binding loop residues in the G94D mutant closely match those in the CA/CypA assembly. Interestingly, earlier-solution NMR studies on an unassembled NTD of the G94D mutant CA indicated no changes in dynamics or conformation with respect to the wild-type CA (26), in contrast with our results. Therefore, it appears that in this mutant the modulation of CypA loop conformation and dynamics requires the presence of the C-terminal domain (CTD) and/or assembly. For the A92E mutant, the S values are higher than those in G94D and CA/CypA, indicating that motions are further restricted. The lineshapes of G89 and A92/E92 are clearly anisotropic with S of 0.21(G89)/0.32(A92) and 0.43(G89)/0.55(E92) for the G94D and A92E mutants, respectively.

The above findings are also illustrated in Fig. 4, where we mapped the N–H dipolar order parameters for all five assemblies onto the 3D structure of an isolated CA protein. Overall, the NMR dipolar parameters indicate (i) a remarkably high degree of motions on nanosecond–microsecond timescales for residues in the CypA-binding loop of wild-type CA from both HXB2 and NL4-3 strains; (ii) a significant decrease in the loop motions in CA/CypA assemblies and the two CypA escape mutants, A92E and G94D; and (iii) similar dynamic profiles for CA/CypA, A92E, and G94D assemblies.

Fig. 4.

Summary of nanosecond to millisecond timescales dynamics observed in tubular assemblies of HIV-1 CA protein. (A) Peak intensities observed in the 2D NCACX MAS NMR correlation spectra plotted vs. residue number (for nonoverlapping peaks with resonance assignments) for tubular assemblies of CA NL4-3 (red), CA NL4-3 A92E (light blue), CA NL4-3 G94D (green), CA HXB2 (black), and CA HXB2/CypA (yellow). The secondary structure is indicated at the bottom: H, helix; S, sheet; L, loop. Note the attenuated peak intensities or missing peaks in CypA loop and throughout the protein, which indicate the presence of motions on microsecond to millisecond timescales. (B) ¹H-¹⁵N S (Upper) and normalized peak intensities (Lower) mapped onto the CA structure for (from left to right): CA HXB2, CA HXB2/CypA, CA NL4-3, CA NL4-3 A92E, and CA NL4-3 G94D.

We next turned our attention to dynamics on the microsecond–millisecond timescales. As reported previously by us, such motions are seen in the hinge region of CA (residues 144–148) and are directly connected to the ability of the CA protein to assemble into pleiomorphic conical capsids (12). Dynamics in this time regime can be detected qualitatively from the peak intensities in the heteronuclear correlation spectra because such motions would interfere with CP, decoupling, or MAS, causing reductions in peak intensity or complete peak disappearance.

Fig. 4 displays normalized peak intensities plotted vs. residue number for all five assemblies measured from the NCA spectra and their mapping onto the 3D structure of a single CA molecule. Missing or weakened signals are noted for many residues, indicating that assembled CA is mobile on this timescale. Consistent with our findings for nanosecond to microsecond dynamics, the loop regions of wild-type CA exhibit significant motions in the microsecond to millisecond regime. In the CA/CypA assemblies, the motions are somewhat attenuated, but, interestingly, CypA-binding loop still remains flexible in this time window. This observation is also made for the CypA-binding loop in the G94D mutant, whereas in the A92E mutant these loop motions are somewhat slower.

More surprisingly, the microsecond–millisecond timescale motions are not restricted to only loops, but occur throughout the entire protein, including the helices of both the CTD and the NTD. Among the five assemblies, motions of the wild-type CA assembly are the fastest on this slow timescale, which, interestingly, are not perturbed significantly upon CypA binding. These motions are likely important for the relative repositioning of the NTD and CTD around the hinge region, associated with the varied curvature in tubular and conical assemblies.

MD Simulations and Calculations of NMR Dipolar Parameters.

The surprising experimental observation of the unusually high extent of dynamics of the CypA-binding loop residues in CA assemblies, which is significantly affected by a single amino acid change, prompted us to scrutinize these results. To that end, we pursued all-atom MD simulations. With the currently available computational resources, we were able to calculate MD trajectories for a single molecule of wild-type CA (HXB2 strain) and for the conical A92E CA mutant in the capsid assembly (NL4-3 strain) to 100 ns (13). These simulations were sufficiently long to recapitulate the experimental NMR dipolar order parameters, as discussed below, but not long enough to evaluate the motions that occur on microsecond–millisecond timescales.

As depicted in Fig. 2, the ¹H-¹⁵N dipolar order parameters computed from the MD trajectories for both wild-type CA and the A92E mutant are in remarkable agreement with the experimental NMR values. Indeed, it is striking that the calculations not only correctly predict the flexible regions, such as CypA-binding loop, but that the calculated S values are also in good quantitative agreement with the experiment. Specifically, the MD simulations capture the experimentally observed differences in the CypA loop dynamics well, substantiating that the loop in wild-type CA assemblies is significantly more flexible than that in the A92E mutant assemblies.

We also performed calculations of dipolar order parameters from multiple MD trajectories for different substructures of capsid assemblies. To that end, we extracted a hexamer of hexamers (HOH) unit from the all atom model of a conical capsid (13). As illustrated in Fig. 2B, the dipolar order parameters calculated for each hexamer in the HOH building block agree well with each other and with the experimental results, indicating the validity of our approach. To our knowledge, this work establishes, for the first time, quantitative agreement between MD simulation-derived and experimental order parameters for such a large assembly.

An important advantage of using the MD simulations is that the trajectories of the CypA-binding loop in the two CA sequence polymorphs can be visualized as angular probability distributions and in 3D scatter plots that depict the bond vector orientations in space. These are plotted in SI Appendix, Fig. S8, for several representative rigid CA residues of CA, as well as for mobile residues in the CypA-binding loop. Clearly, the remarkable mobility of the CypA-binding loop in wild-type CA assemblies is associated with these loop residues sampling a large portion of the available 3D space. Interestingly, the corresponding angular probability distributions do not exhibit a canonical Gaussian shape and may even possess two local maxima, such as seen for residue G94.

Discussion

The effect of CypA on the HIV-1 infection is complex and poorly understood. In humans, the HIV-1 infectivity is enhanced by the interaction of CypA with the capsid (29, 30). On the contrary, in other primates, CypA interferes with the viral infection by enhancing the activity of the restriction factors TRIM5α and TRIM-Cyp (31, 32). Moreover, depending on the cell type, the capsid can be either stabilized or destabilized by CypA (5, 6). Escape mutations in the CypA loop result in the loss of 90% of the HIV-1 infectivity in the presence of CypA; the activity is restored upon CypA inhibition (8).

Changes in the overall CA structures due to mutations are unlikely the explanation for the escape from CypA dependence: Given the relatively modest chemical shift differences, most of which are limited to CypA loop residues, we are confident that the 3D structures of the wild-type CA and the two CypA escape mutants are very similar. Furthermore, the binding affinities of CypA to the monomeric CypA escape mutants do not differ much from those to the wild-type CA (8). We therefore suggest that the mechanism of capsid’s escape from CypA dependence involves a change in dynamics.

The NMR and MD simulation data reveal an unequivocal relationship between the dynamic profile of CA and the mutants. Wild-type CA of the HXB2 and NL4-3 strains is remarkably flexible in the assembled state. In particular, the extent of motional averaging seen for CypA-binding loop residues appears to be unprecedented and, to our knowledge, has not been seen in any other proteins that have been investigated by MAS NMR. Binding of CypA quenches the motions in the CypA loop, as well as of many other residues in the protein.

To our surprise, the two CypA escape mutants exhibited dynamic signatures very similar to that of wild-type CA in complex with CypA. Specifically, the CypA-binding loop dynamics in the A92E and G94D mutants resembles that of the CA/CypA complex. Remarkably, MD simulations of wild-type CA and the A92E mutant near quantitatively recapitulate the experimentally derived NMR dipolar tensor parameters, suggesting that CypA escape is intimately coupled to dynamics.

We further propose that dynamic allostery is the main mechanism for regulation of viral infectivity, providing a more subtle way for fine-tuning activity than would result from an overall change in the structure of the protein. In fact, dynamic allostery as a regulatory mechanism is commonly used in many biological processes (33), including, but not limited to, receptor activation in signaling (34), enzymatic catalysis (35, 36) including viral maturation involving proteolysis (37), intracellular transport (38–40), and others.

Concluding Remarks

Using MAS NMR experiments and MD simulations, we investigated conformational dynamics in HIV-1 CA protein assemblies. We discovered that CA in the assembled state is highly mobile on the nanosecond to millisecond timescales. A surprising finding was that the motional signatures of residues in the CypA-binding loop of CypA escape mutants are similar to those in CA/CypA assemblies, suggesting a previously unidentified mechanism for CypA escape by dynamic allostery. New therapeutic intervention strategies may be envisioned through modulation of CypA-binding loop dynamics by small-molecule interactors. Finally, our MAS NMR and MD approach is also applicable for the analysis of CA assemblies with other host factors.

Materials and Methods

CA (HXB2, NL4-3, NL4-3/A92E, and NL4-3/G94D variants) and CypA were expressed and purified by using the same protocol as reported (14, 41). Tubular assemblies of CA protein were prepared and their morphology characterized as described (14) and detailed in SI Appendix, SI Text. Details of MAS NMR experiments, MD simulations, and spectral and dynamics analysis are described in SI Appendix, SI Text.