Structure of the N-terminal 283-residue fragment of the immature HIV-1 Gag polyprotein

The capsid protein (CA) of the mature human immunodeficiency virus (HIV) contains an N-terminal beta-hairpin that is essential for formation of the capsid core particle. CA is generated by proteolytic cleavage of the Gag precursor polyprotein during viral maturation. We have determined the NMR structure of a 283-residue N-terminal fragment of immature HIV-1 Gag (Gag(283)), which includes the intact matrix (MA) and N-terminal capsid (CA(N)) domains. The beta-hairpin is unfolded in Gag(283), consistent with the proposal that hairpin formation occurs subsequent to proteolytic cleavage of Gag, triggering capsid assembly. Comparison of the immature and mature CA(N) structures reveals that beta-hairpin formation induces a approximately 2 A displacement of helix 6 and a concomitant displacement of the cyclophylin-A (CypA)-binding loop, suggesting a possible allosteric mechanism for CypA-mediated destabilization of the capsid particle during infectivity.     

Catalysis and binding of cyclophilin A with different HIV-1 capsid constructs

The prolyl isomerase cyclophilin A (CypA) is required for efficient HIV-1 replication and is incorporated into virions through a binding interaction at the Gly-Pro(222) bond located within the capsid domain of the HIV-1 Gag precursor polyprotein (Pr(gag)). It has recently been shown that CypA efficiently catalyzes the cis/trans isomerization of Gly-Pro(222) within the isolated N-terminal domain of capsid (CA(N)). To address the proposal that CypA interacts with Gly-Pro sequences in the C-terminal domain of a mature capsid, the interaction between CypA and the natively folded, full-length capsid protein (CA(FL)) has been investigated here using nuclear magnetic resonance spectroscopy. In addition, a fragment of the Pr(gag) protein encoding the full-matrix protein and the N-terminal domain of capsid (MA-CA(N)) has been used to probe the catalytic interaction between CypA and an immature form of the capsid. The results discussed herein strongly suggest that Gly-Pro(222) located within the N-terminal domain of the capsid is the preferential site for CypA binding and catalysis and that catalysis of Gly-Pro(222) is unaffected by maturational processing at the N-terminus of the capsid.     

Altered HIV-1 Gag protein interactions with cyclophilin A (CypA) on the acquisition of H219Q and H219P substitutions in the CypA binding loop

HIV-1 Gag protein interaction with cyclophilin A (CypA) is critical for viral fitness. Among the amino acid substitutions identified in Gag noncleavage sites in HIV-1 variants resistant to protease inhibitors, H219Q (Gatanaga, H., Suzuki, Y., Tsang, H., Yoshimura, K., Kavlick, M. F., Nagashima, K., Gorelick, R. J., Mardy, S., Tang, C., Summers, M. F., and Mitsuya, H. (2002) J. Biol. Chem. 277, 5952-5961) and H219P substitutions in the viral CypA binding loop confer the greatest replication advantage to HIV-1. These substitutions represent polymorphic amino acid residues. We found that the replication advantage conferred by these substitutions was far greater in CypA-rich MT-2 and H9 cells than in Jurkat cells and peripheral blood mononuclear cells (PBM), both of which contained less CypA. High intracellular CypA content in H9 and MT-2 cells, resulting in excessive CypA levels in virions, limited wild-type HIV-1 (HIV-1(WT)) replication and H219Q introduction into HIV-1 (HIV-1(H219Q)), reduced CypA incorporation of HIV-1, and potentiated viral replication. H219Q introduction also restored the otherwise compromised replication of HIV-1(P222A) in PBM, although the CypA content in HIV-1(H219Q/P222A) was comparable with that in HIV-1(P222A), suggesting that H219Q affected the conformation of the CypA-binding motif, rendering HIV-1 replicative in a low CypA environment. Structural modeling analyses revealed that although hydrogen bonds are lost with H219Q and H219P substitutions, no significant distortion of the CypA binding loop of Gag occurred. The loop conformation of HIV-1(P222A) was found highly distorted, although H219Q introduction to HIV-1 restored the conformation of the loop close to that of HIV-1 (P222A). The present data suggested that the effect of CypA on HIV-1 replicative (WT) ability is bimodal (both high and low CypA content limits HIV-1 replication), that the conformation of the CypA binding region of Gag is important for viral fitness, and that the function of CypA is to maintain the conformation.     

The cysteine residues of HIV-1 capsid regulate oligomerization and cyclophilin A-induced changes

Assembly of the HIV-1 virus involves, in part, strong interactions between the capsid (CA) domains of the Gag polyprotein. During maturation, the core of HIV-1 virions undergoes profound morphological changes due primarily to proteolysis of the CA domain from other Gag domains which may allow for more efficient disassembly of the viral core in the early stages of infection. The host protein cyclophilin A (CypA), a cis-trans prolyl isomerase, in some way seems to assist in this assembly/disassembly process. Using an unproteolyzed construct of CA, we show that binding of CypA induces a large-scale conformational change in CA that is independent of its cis-trans prolyl isomerase activity. This change appears to be mediated by Cys-198 of CA since mutation to Ala renders CypA unable to induce this change and alters the kinetics and stability of protein cores that may ultimately result in inefficient disassembly of viral cores. Alternately, mutation of the second CA Cys (C218A) allows for CypA-induced conformational changes but alters the kinetics and morphology of the protein cores that may ultimately result in inefficient assembly of viral cores. These studies show the importance of the CA Cys residues in mediating the contacts needed for viral assembly and disassembly.     

A mutation in alpha helix 3 of CA renders human immunodeficiency virus type 1 cyclosporin A resistant and dependent: rescue by a second-site substitution in a distal region of CA

The replication of many isolates of human immunodeficiency virus type 1 (HIV-1) is enhanced by binding of the host cell protein cyclophilin A (CypA) to the viral capsid protein (CA). The immunosuppressive drug cyclosporine A (CsA) and its nonimmunosuppressive analogs bind with high affinity to CypA and inhibit HIV-1 replication. Previous studies have identified two mutations, A92E and G94D, in the CypA-binding loop of CA that confer the ability of HIV-1 to replicate in the presence of CsA. Interestingly, CsA stimulates the replication of HIV-1 mutants containing either the A92E or G94D substitution in some human cell lines. Here, we show that substitution of alanine for threonine at position 54 of CA (T54A) also confers HIV-1 resistance to and dependence on CsA. Like the previously identified CsA-resistant/dependent mutants, infection by the T54A mutant was stimulated by CsA in a target cell-specific manner. RNA interference-mediated reduction of CypA expression enhanced the permissiveness of HeLa cells to infection by the T54A mutant. A suppressor mutation, encoding a substitution of threonine for alanine at position 105 of CA (A105T), was identified through adaptation of the T54A mutant virus for growth in CEM cells. A105T rescued the impaired single-cycle infectivity and replication defects of both T54A and A92E mutants. These results indicate that CA determinants outside the CypA-binding loop can modulate the dependence of HIV-1 infection on CypA.     

trans-Complementation rescue of cyclophilin A-deficient viruses reveals that the requirement for cyclophilin A in human immunodeficiency virus type 1 replication is independent of its isomerase activity

Human immunodeficiency virus type 1 (HIV-1) requires the incorporation of cyclophilin A (CypA) for replication. CypA is packaged by binding to the capsid (CA) region of Gag. This interaction is disrupted by cyclosporine (CsA). Preventing CypA incorporation, either by mutations in the binding region of CA or by the presence of CsA, abrogates virus infectivity. Given that CypA possesses an isomerase activity, it has been proposed that CypA acts as an uncoating factor by destabilizing the shell of CA that surrounds the viral genome. However, because the same domain of CypA is responsible for both its isomerase activity and its capacity to be packaged, it has been challenging to determine if isomerase activity is required for HIV-1 replication. To address this issue, we fused CypA to viral protein R (Vpr), creating a Vpr-CypA chimera. Because Vpr is packaged via the p6 region of Gag, this approach bypasses the interaction with CA and allows CypA incorporation even in the presence of CsA. Using this system, we found that Vpr-CypA rescues the infectivity of viruses lacking CypA, either produced in the presence of CsA or mutated in the CypA packaging signal of CA. Furthermore, a Vpr-CypA mutant which has no isomerase activity and no capacity to bind to CA also rescues HIV-1 replication. Thus, this study demonstrates that the isomerase activity of CypA is not required for HIV-1 replication and suggests that the interaction of the catalytic site of CypA with CA serves no other function than to incorporate CypA into viruses.     

Hydrogen/deuterium exchange analysis of HIV-1 capsid assembly and maturation

Following budding, HIV-1 virions undergo a maturation process where the Gag polyprotein in the immature virus is cleaved by the viral protease and rearranges to form the mature infectious virion. Despite the wealth of structures of isolated capsid domains and an in?vitro-assembled mature lattice, models of the immature lattice do not provide an unambiguous model of capsid-molecule orientation and no structural information is available for the capsid maturation pathway. Here we have applied hydrogen/deuterium exchange mass spectrometry to immature, mature, and mutant Gag particles (CA5) blocked at the final Gag cleavage event to examine the molecular basis of capsid assembly and maturation. Capsid packing arrangements were very similar for all virions, whereas immature and CA5 virions contained an additional intermolecular interaction at the hexameric, 3-fold axis. Additionally, the N-terminal β-hairpin was observed to form as a result of capsid-SP1 cleavage rather than driving maturation as previously postulated.     

Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity

The peptidyl-prolyl isomerase cyclophilin A (CypA) increases the kinetics by which human immunodeficiency virus type 1 (HIV-1) spreads in tissue culture. This was conclusively demonstrated by gene targeting in human CD4(+) T cells, but the role of CypA in HIV-1 replication remains unknown. Though CypA binds to mature HIV-1 capsid protein (CA), it is also incorporated into nascent HIV-1 virions via interaction with the CA domain of the Gag polyprotein. These findings raised the possibility that CypA might act at multiple steps of the retroviral life cycle. Disruption of the CA-CypA interaction, either by the competitive inhibitor cyclosporine (CsA) or by mutation of CA residue G89 or P90, suggested that producer cell CypA was required for full virion infectivity. However, recent studies indicate that CypA within the target cell regulates HIV-1 infectivity by modulating Ref1- or Lv1-mediated restriction. To examine the relative contribution to HIV-1 replication of producer cell CypA and target cell CypA, we exploited multiple tools that disrupt the HIV-1 CA-CypA interaction. These tools included the drugs CsA, MeIle(4)-CsA, and Sanglifehrin; CA mutants exhibiting decreased affinity for CypA or altered CypA dependence; HeLa cells with CypA knockdown by RNA interference; and Jurkat T cells homozygous for a deletion of the gene encoding CypA. Our results clearly demonstrate that target cell CypA, and not producer cell CypA, is important for HIV-1 CA-mediated function. Inhibition of HIV-1 infectivity resulting from virion production in the presence of CsA occurs independently of the CA-CypA interaction or even of CypA.     

Flexibility in the P2 domain of the HIV-1 Gag polyprotein

The HIV-1 Gag polyprotein contains a segment called p2, located between the capsid (CA) and nucleocapsid (NC) domains, that is essential for ordered virus assembly and infectivity. We subcloned, overexpressed, and purified a 156-residue polypeptide that contains the C-terminal capsid subdomain (CA(CTD)) through the NC domain of Gag (CA(CTD)-p2-NC, Gag residues 276-431) for NMR relaxation and sedimentation equilibrium (SE) studies. The CA(CTD) and NC domains are folded as expected, but residues of the p2 segment, and the adjoining thirteen C-terminal residues of CA(CTD) and thirteen N-terminal residues of NC, are flexible. Backbone NMR chemical shifts of these 40 residues deviate slightly from random coil values and indicate a small propensity toward an alpha-helical conformation. The presence of a transient coil-to-helix equilibrium may explain the unusual and necessarily slow proteolysis rate of the CA-p2 junction. CA(CTD)-p2-NC forms dimers and self-associates with an equilibrium constant (Kd = 1.78 +/- 0.5 microM) similar to that observed for the intact capsid protein (Kd = 2.94 +/- 0.8 microM), suggesting that Gag self-association is not significantly influence by the P2 domain.     

Analysis of human immunodeficiency virus type 1 Gag dimerization-induced assembly

The nucleocapsid (NC) domains of retrovirus precursor Gag (PrGag) proteins play an essential role in virus assembly. Evidence suggests that NC binding to viral RNA promotes dimerization of PrGag capsid (CA) domains, which triggers assembly of CA N-terminal domains (NTDs) into hexamer rings that are interconnected by CA C-terminal domains. To examine the influence of dimerization on human immunodeficiency virus type 1 (HIV-1) Gag protein assembly in vitro, we analyzed the assembly properties of Gag proteins in which NC domains were replaced with cysteine residues that could be linked via chemical treatment. In accordance with the model that Gag protein pairing triggers assembly, we found that cysteine cross-linking or oxidation reagents induced the assembly of virus-like particles. However, efficient assembly also was observed to be temperature dependent or required the tethering of NTDs. Our results suggest a multistep pathway for HIV-1 Gag protein assembly. In the first step, Gag protein pairing through NC-RNA interactions or C-terminal cysteine linkage fosters dimerization. Next, a conformational change converts assembly-restricted dimers or small oligomers into assembly-competent ones. At the final stage, final particle assembly occurs, possibly through a set of larger intermediates.     

Protease Cleavage Leads to Formation of Mature Trimer Interface in HIV-1 Capsid

During retrovirus particle maturation, the assembled Gag polyprotein is cleaved by the viral protease into matrix (MA), capsid (CA), and nucleocapsid (NC) proteins. To form the mature viral capsid, CA rearranges, resulting in a lattice composed of hexameric and pentameric CA units. Recent structural studies of assembled HIV-1 CA revealed several inter-subunit interfaces in the capsid lattice, including a three-fold interhexamer interface that is critical for proper capsid stability. Although a general architecture of immature particles has been provided by cryo-electron tomographic studies, the structural details of the immature particle and the maturation pathway remain unknown. Here, we used cryo-electron microscopy (cryoEM) to determine the structure of tubular assemblies of the HIV-1 CA-SP1-NC protein. Relative to the mature assembled CA structure, we observed a marked conformational difference in the position of the CA-CTD relative to the NTD in the CA-SP1-NC assembly, involving the flexible hinge connecting the two domains. This difference was verified via engineered disulfide crosslinking, revealing that inter-hexamer contacts, in particular those at the pseudo three-fold axis, are altered in the CA-SP1-NC assemblies compared to the CA assemblies. Results from crosslinking analyses of mature and immature HIV-1 particles containing the same Cys substitutions in the Gag protein are consistent with these findings. We further show that cleavage of preassembled CA-SP1-NC by HIV-1 protease in vitro leads to release of SP1 and NC without disassembly of the lattice. Collectively, our results indicate that the proteolytic cleavage of Gag leads to a structural reorganization of the polypeptide and creates the three-fold interhexamer interface, important for the formation of infectious HIV-1 particles.     

Human immunodeficiency virus type 1 (HIV-1) protein Vif inhibits the activity of HIV-1 protease in bacteria and in vitro.

Human immunodeficiency virus type 1 (HIV-1) Vif is required for productive infection of T lymphocytes and macrophages. Virions produced in the absence of Vif have abnormal core morphology and those produced in primary T cells carry immature core proteins and low levels of mature capsid (M. Simm, M. Shahabuddin, W. Chao, J. S. Allan, and D. J. Volsky, J. Virol. 69:4582-4586, 1995). To investigate whether Vif influences the activity of HIV-1 protease (PR), the viral enzyme which is responsible for processing Gag and Gag-Pol precursor polyproteins into mature virion components, we transformed bacteria to inducibly express truncated Gag-Pol fusion proteins and Vif. We examined the cleavage of polyproteins consisting of matrix to PR (Gag-PR), capsid to PR (CA-PR), and p6Pol to PR (p6Pol-PR) and evaluated HIV-1 protein processing at specific sites by Western blotting using antibodies against matrix, capsid, and PR proteins. We found that Vif modulates HIV-1 PR activity in bacteria mainly by preventing the release of mature MA and CA from Gag-PR, CA from CA-PR, and p6Pol from p6Pol-PR, with other cleavages being less affected. Using subconstructs of Vif, we mapped this activity to the N-terminal half of the molecule, thus identifying a new functional domain of Vif. Kinetic study of p6Pol-PR autocatalysis in the presence or absence of Vif revealed that Vif and N'Vif reduce the rate of PR-mediated proteolysis of this substrate. In an assay of in vitro proteolysis of a synthetic peptide substrate by purified recombinant PR we found that recombinant Vif and the N-terminal half of the molecule specifically inhibit PR activity at a molar ratio of the N-terminal half of Vif to PR of about 1. These results suggest a mechanism and site of action of Vif in HIV-1 replication and demonstrate novel regulation of a lentivirus PR by an autologous viral protein acting in trans.     

Structural analysis of the N-terminal domain of the human T-cell leukemia virus capsid protein

The N-terminal domain of the retroviral capsid (CA) protein is one of the least conserved regions encoded in the genome. Surprisingly, the three-dimensional structures of the CA from different genera exhibit alpha-helical structural features that are highly conserved. The N-terminal residues of the human immunodeficiency virus type 1 (HIV-1) and Rous sarcoma virus (RSV) capsid proteins form a beta-hairpin. To determine if this feature is conserved in the retroviral family, we cloned, expressed, purified, and solved the structure of a N-terminal 134 amino acid fragment (CA(134)) from the human T-cell leukemia virus type 1 (HTLV-I) using high resolution nuclear magnetic resonance (NMR) spectroscopy. The CA(134) fragment contains an N-terminal beta-hairpin and a central coiled-coil-like structure composed of six alpha-helices. The N-terminal Pro1 residue contacts Asp54 in the helical cluster through a salt bridge. Thus, the beta-hairpin is conserved and the helical cluster is structurally similar to other retroviral CA domains. However, although the same Asp residue defines the orientation of the hairpin in both the HTLV-1 and HIV-1 CA proteins, the HTLV-I hairpin is oriented away, rather than towards, the helical core. Significant differences were also detected in the spatial orientation and helical content of the long centrally located loop connecting the helices in the core. It has been proposed that the salt bridge allows the formation of a CA-CA interface that is important for the assembly of the conical cores that are characteristic of HIV-1. As HTLV-I forms spherical cores, the salt-bridge feature is apparently not conserved for this function although its role in determining the orientation of the beta-hairpin may be critical, along with the central loop. Comparison of three-dimensional structures is expected to elucidate the relationships between the retroviral capsid protein structure and its function.     

Antiviral activity of recombinant ankyrin targeted to the capsid domain of HIV-1 Gag polyprotein

Background

Ankyrins are cellular mediators of a number of essential protein-protein interactions. Unlike intrabodies, ankyrins are composed of highly structured repeat modules characterized by disulfide bridge-independent folding. Artificial ankyrin molecules, designed to target viral components, might act as intracellular antiviral agents and contribute to the cellular immunity against viral pathogens such as HIV-1.

Results

A phage-displayed library of artificial ankyrins was constructed, and screened on a polyprotein made of the fused matrix and capsid domains (MA-CA) of the HIV-1 Gag precursor. An ankyrin with three modules named Ank^GAG1D4 (16.5 kDa) was isolated. Ank^GAG1D4 and MA-CA formed a protein complex with a stoichiometry of 1:1 and a dissociation constant of K _d ~ 1 ??M, and the Ank^GAG1D4 binding site was mapped to the N-terminal domain of the CA, within residues 1-110. HIV-1 production in SupT1 cells stably expressing Ank^GAG1D4 in both N-myristoylated and non-N-myristoylated versions was significantly reduced compared to control cells. Ank^GAG1D4 expression also reduced the production of MLV, a phylogenetically distant retrovirus. The Ank^GAG1D4-mediated antiviral effect on HIV-1 was found to occur at post-integration steps, but did not involve the Gag precursor processing or cellular trafficking. Our data suggested that the lower HIV-1 progeny yields resulted from the negative interference of Ank^GAG1D4-CA with the Gag assembly and budding pathway.

Conclusions

The resistance of Ank^GAG1D4-expressing cells to HIV-1 suggested that the CA-targeted ankyrin Ank^GAG1D4 could serve as a protein platform for the design of a novel class of intracellular inhibitors of HIV-1 assembly based on ankyrin-repeat modules.     

Species-specific tropism determinants in the human immunodeficiency virus type 1 capsid

Retroviral tropism is determined in part by cellular restriction factors that block infection by targeting the incoming viral capsid. Indeed, human immunodeficiency virus type 1 (HIV-1) infection of many nonhuman primate cells is inhibited by one such factor, termed Lv1. In contrast, a restriction factor in humans, termed Ref1, does not inhibit HIV-1 infection unless nonnatural mutations are introduced into the HIV-1 capsid protein (CA). Here, we examined the infectivity of a panel of mutant HIV-1 strains carrying substitutions in the N-terminal CA domain in cells that exhibit restriction attributable to Lv1 or Ref1. Manipulation of HIV-1 CA could alter HIV-1 tropism, and several mutations were identified that increased or decreased HIV-1 infectivity in a target-cell-specific manner. Many residues that affected HIV-1 tropism were located in the three variable loops that lie on the outer surface of the modeled HIV-1 conical capsid. Some tropism determinants, including the CypA binding site, coincided with residues whose mutation conferred on HIV-1 CA the ability to saturate Ref1 in human cells. Notably, a mutation that reverses the infectivity defect in human cells induced by CypA binding site mutation inhibits recognition by Ref1. Overall, these findings demonstrate that exposed variable loops in CA and a partial CypA "coat" can modulate restriction and HIV-1 tropism and suggest a model in which the exposed surface of the incoming retroviral capsid is the target for inhibition by host cell-specific restriction factors.     

Naturally occurring capsid substitutions render HIV-1 cyclophilin A independent in human cells and TRIM-cyclophilin-resistant in Owl monkey cells

In this study, we asked if a naturally occurring HIV-1 variant exists that circumvents CypA dependence in human cells. To address this issue, we sought viruses for CypA independence using Debio-025, a cyclosporine A (CsA) analog that disrupts CypA-capsid interaction. Surprisingly, viral variants from the Main group replicate even in the presence of the drug. Sequencing analyses revealed that these viruses encode capsid substitutions within the CypA-binding site (V86P/H87Q/I91V/M96I). When we introduced these substitutions into viruses that normally rely on CypA for replication, these mutants no longer depended on CypA, suggesting that naturally occurring capsid substitutions obviate the need for CypA. This is the first demonstration that isolates from the Main group naturally develop CypA-independent strategies to replicate in human cells. Surprisingly, we found that these capsid substitutions render HIV-1 capable of infecting Owl monkey (OMK) cells that highly restrict HIV-1. OMK cell resistance to HIV-1 is mediated via TRIM-Cyp, which arose from a retrotransposition of CypA into the TRIM5 alpha gene. Interestingly, saturation experiments suggest that the Pro86/Gln87/Val91/Ile96 capsid core is "invisible" to TRIM-Cyp. This study demonstrates that specific capsid substitutions can release HIV-1 from both CypA dependence in human cells and TRIM-Cyp restriction in monkey cells.     

Escape and compensation from early HLA-B57-mediated cytotoxic T-lymphocyte pressure on human immunodeficiency virus type 1 Gag alter capsid interactions with cyclophilin A

Certain histocompatibility leukocyte antigen (HLA) alleles are associated with improved clinical outcomes for individuals infected with human immunodeficiency virus type 1 (HIV-1), but the mechanisms for their effects remain undefined. An early CD8(+) T-cell escape mutation in the dominant HLA-B57-restricted Gag epitope TW10 (TSTLQEQIGW) has been shown to impair HIV-1 replication capacity in vitro. We demonstrate here that this T(242)N substitution in the capsid protein is associated with upstream mutations at residues H(219), I(223), and M(228) in the cyclophilin A (CypA)-binding loop in B57(+) individuals with progressive disease. In an independent cohort of epidemiologically linked transmission pairs, the presence of these substitutions in viruses encoding T(242)N was associated with significantly higher plasma viremia in donors, further suggesting that these secondary mutations compensated for the replication defect of T(242)N. Using NL4-3 constructs, we illustrate the ability of these CypA loop changes to partially restore replication of the T(242)N variant in vitro. Notably, these mutations also enhanced viral resistance to the drug cyclosporine A, indicating a reduced dependence of the compensated virus on CypA that is normally essential for optimal infectivity. Therefore, mutations in TW10 allow HIV-1 to evade a dominant early CD8(+) T-cell response, but the benefits of escape are offset by a defect in capsid function. These data suggest that TW10 escape variants undergo a postentry block that is partially overcome by changes in the CypA-binding loop and identify a mechanism for an HIV-1 fitness defect that may contribute to the slower disease progression associated with HLA-B57.     

Cis/trans isomerization in HIV-1 capsid protein catalyzed by cyclophilin A: insights from computational and theoretical studies

A network of protein vibrations has recently been identified in the enzyme cyclophilin A (CypA) that is associated with its peptidyl-prolyl cis/trans isomerization activity of small peptide substrates. It has been suggested that this network may have a role in promoting the catalytic step during the isomerization reaction. This work presents the results from the characterization of this network during the isomerization of the Gly89-Pro90 peptide bond in the N-terminal domain of the capsid protein (CA(N)) from human immunodeficiency virus type 1 (HIV-1), which is a naturally occurring, biologically relevant protein substrate for CypA. A variety of computational and theoretical studies are utilized to investigate the protein dynamics of the CypA-CA(N) complex, at multiple time scales, during the isomerization step. The results provide insights into the detailed mechanism of isomerization and confirm the presence of previously reported network of protein vibrations coupled to the reaction. Conserved CypA residues at the complex interface and at positions distal to the interface form parts of this network. There is HIV-1 related medical interest in CypA; incorporation of CypA, complexed with the capsid protein, into the virion is required for the infectious activity of HIV-1. Interaction energy and dynamical cross-correlation calculations are used for a detailed investigation of the protein-protein interactions in the CypA-CA(N) complex. The results show that CA(N) residues His87-Ala-Gly-Pro-Ile-Ala92 form the majority of the interactions with CypA residues. New protein-protein interactions distal to the active site (CypA Arg148-CA(N) Gln95 and CypA Arg148-CA(N) Asn121) are also identified.