Annie Aniana

Visualizing transient events in amino-terminal autoprocessing of HIV-1 protease.

HIV-1 protease processes the Gag and Gag-Pol polyproteins into mature structural and functional proteins, including itself, and is therefore indispensable for viral maturation. The mature protease is active only as a dimer with each subunit contributing catalytic residues. The full-length transframe region protease precursor appears to be monomeric yet undergoes maturation via intramolecular cleavage of a putative precursor dimer, concomitant with the appearance of mature-like catalytic activity. How such intramolecular cleavage can occur when the amino and carboxy termini of the mature protease are part of an intersubunit β-sheet located distal from the active site is unclear. Here we visualize the early events in N-terminal autoprocessing using an inactive mini-precursor with a four-residue N-terminal extension that mimics the transframe region protease precursor. Using paramagnetic relaxation enhancement, a technique that is exquisitely sensitive to the presence of minor species, we show that the mini-precursor forms highly transient, lowly populated (3–5%) dimeric encounter complexes that involve the mature dimer interface but occupy a wide range of subunit orientations relative to the mature dimer. Furthermore, the occupancy of the mature dimer configuration constitutes a very small fraction of the self-associated species (accounting for the very low enzymatic activity of the protease precursor), and the N-terminal extension makes transient intra- and intersubunit contacts with the substrate binding site and is therefore available for autocleavage when the correct dimer orientation is sampled within the encounter complex ensemble.

Inhibition of autoprocessing of natural variants and multidrug resistant mutant precursors of HIV-1 protease by clinical inhibitors.

Self-cleavage at the N terminus of HIV-1 protease from the Gag-Pol precursor (autoprocessing) is crucial for stabilizing the protease dimer required for onset of mature-like catalytic activity, viral maturation, and propagation. Among nine clinical protease inhibitors (PIs), darunavir and saquinavir were the most effective in inhibiting wild-type HIV-1 group M precursor autoprocessing, with an IC50 value of 1–2 μM, 3–5 orders of magnitude higher than their binding affinities to the corresponding mature protease. Accordingly, both group M and N precursor–PI complexes exhibit Tms 17–21 °C lower than those of the corresponding mature protease–PI complexes suggestive of markedly reduced stabilities of the precursor dimer–PI ensembles. Autoprocessing of group N (natural variant) and three group M precursors bearing 11–20 mutations associated with multidrug resistance was either weakly responsive or fully unresponsive to inhibitors at concentrations up to a practical limit of approximately 150 μM PI. This observation parallels decreases of up to 8 × 103-fold (e.g., 5 pM to 40 nM) in the binding affinity of darunavir and saquinavir to mature multidrug resistant proteases relative to wild type, suggesting that inhibition of some of these mutant precursors will occur only in the high μM to mM range in extreme PI-resistance, which is an effect arising from coordinated multiple mutations. An extremely darunavir-resistant mutant precursor is more responsive to inhibition by saquinavir. These findings raise the questions whether clinical failure of PI therapy is related to lack of inhibition of autoprocessing and whether specific inhibitors can be designed with low-nM affinity to target autoprocessing.

Revealing the dimer dissociation and existence of a folded monomer of the mature HIV‐2 protease

Purification and in vitro protein‐folding schemes were developed to produce monodisperse samples of the mature wild‐type HIV‐2 protease (PR2), enabling a comprehensive set of biochemical and biophysical studies to assess the dissociation of the dimeric protease. An E37K substitution in PR2 significantly retards autoproteolytic cleavage during expression. Furthermore, it permits convenient measurement of the dimer dissociation of PR2E37K (elevated Kd ∼20 nM) by enzyme kinetics. Differential scanning calorimetry reveals a Tm of 60.5 for PR2 as compared with 65.7°C for HIV‐1 protease (PR1). Consistent with weaker binding of the clinical inhibitor darunavir (DRV) to PR2, the Tm of PR2 increases by 14.8°C in the presence of DRV as compared with 22.4°C for PR1. Dimer interface mutations, such as a T26A substitution in the active site (PR2T26A) or a deletion of the C‐terminal residues 96–99 (PR21–95), drastically increase the Kd (>105‐fold). PR2T26A and PR21–95 consist predominantly of folded monomers, as determined by nuclear magnetic resonance (NMR) and size‐exclusion chromatography coupled with multiangle light scattering and refractive index measurements (SMR), whereas wild‐type PR2 and its active‐site mutant PR2D25N are folded dimers. Addition of twofold excess active‐site inhibitor promotes dimerization of PR2T26A but not of PR21–95, indicating that subunit interactions involving the C‐terminal residues are crucial for dimer formation. Use of SMR and NMR with PR2 facilitates probing for potential inhibitors that restrict protein folding and/or dimerization and, thus, may provide insights for the future design of inhibitors to circumvent drug resistance.

Complete dissociation of the HIV-1 gp41 ectodomain and membrane proximal regions upon phospholipid binding

The envelope glycoprotein gp41 mediates the process of membrane fusion that enables entry of the HIV-1 virus into the host cell. Strong lipid affinity of the ectodomain suggests that its heptad repeat regions play an active role in destabilizing membranes by directly binding to the lipid bilayers and thereby lowering the free-energy barrier for membrane fusion. In such a model, immediately following the shedding of gp120, the N-heptad and C-heptad helices dissociate and melt into the host cell and viral membranes, respectively, pulling the destabilized membranes into juxtaposition, ready for fusion. Post-fusion, reaching the final 6-helix bundle (6HB) conformation then involves competition between intermolecular interactions needed for formation of the symmetric 6HB trimer and the membrane affinity of gp41’s ectodomain, including its membrane-proximal regions. Our solution NMR study of the structural and dynamic properties of three constructs containing the ectodomain of gp41 with and without its membrane-proximal regions suggests that these segments do not form inter-helical interactions until the very late steps of the fusion process. Interactions between the polar termini of the heptad regions, which are not associating with the lipid surface, therefore may constitute the main driving force initiating formation of the final post-fusion states. The absence of significant intermolecular ectodomain interactions in the presence of dodecyl phosphocholine highlights the importance of trimerization of gp41’s transmembrane helix to prevent complete dissociation of the trimer during the course of fusion.

Binding of Clinical Inhibitors to a Model Precursor of a Rationally Selected Multidrug Resistant HIV-1 Protease Is Significantly Weaker Than That to the Released Mature Enzyme

We have systematically validated the activity and inhibition of a HIV-1 protease (PR) variant bearing 17 mutations (PR(S17)), selected to represent high resistance by machine learning on genotype-phenotype data. Three of five mutations in PR(S17) correlating with major drug resistance, M46L, G48V, and V82S, and five of 11 natural variations differ from the mutations in two clinically derived extreme mutants, PR20 and PR22 bearing 19 and 22 mutations, respectively. PR(S17), which forms a stable dimer (<10 nM), is ∼10- and 2-fold less efficient in processing the Gag polyprotein than the wild type and PR20, respectively, but maintains the same cleavage order. Isolation of a model precursor of PR(S17) flanked by the 56-amino acid transframe region (TFP-p6pol) at its N-terminus, which is impossible upon expression of an analogous PR20 precursor, allowed systematic comparison of inhibition of TFP-p6pol-PR(S17) and mature PR(S17). Resistance of PR(S17) to eight protease inhibitors (PIs) relative to PR (Ki) increases by 1.5-5 orders of magnitude from 0.01 to 8.4 μM. Amprenavir, darunavir, atazanavir, and lopinavir, the most effective of the eight PIs, inhibit precursor autoprocessing at the p6pol/PR site with IC50 values ranging from ∼7.5 to 60 μM. Thus, this process, crucial for stable dimer formation, shows inhibition ∼200-800-fold weaker than that of the mature PR(S17). TFP/p6pol cleavage, which occurs faster, is inhibited even more weakly by all PIs except darunavir (IC50 = 15 μM); amprenavir shows a 2-fold increase in IC50 (∼15 μM), and atazanavir and lopinavir show increased IC50 values of >42 and >70 μM, respectively.

Mutations Proximal to Sites of Autoproteolysis and the α-Helix That Co-evolve under Drug Pressure Modulate the Autoprocessing and Vitality of HIV-1 Protease.

N-Terminal self-cleavage (autoprocessing) of the HIV-1 protease precursor is crucial for liberating the active dimer. Under drug pressure, evolving mutations are predicted to modulate autoprocessing, and the reduced catalytic activity of the mature protease (PR) is likely compensated by enhanced conformational/dimer stability and reduced susceptibility to self-degradation (autoproteolysis). One such highly evolved, multidrug resistant protease, PR20, bears 19 mutations contiguous to sites of autoproteolysis in retroviral proteases, namely clusters 1-3 comprising residues 30-37, 60-67, and 88-95, respectively, accounting for 11 of the 19 mutations. By systematically replacing corresponding clusters in PR with those of PR20, and vice versa, we assess their influence on the properties mentioned above and observe no strict correlation. A 10-35-fold decrease in the cleavage efficiency of peptide substrates by PR20, relative to PR, is reflected by an only ∼4-fold decrease in the rate of Gag processing with no change in cleavage order. Importantly, optimal N-terminal autoprocessing requires all 19 PR20 mutations as evaluated in vitro using the model precursor TFR-PR20 in which PR is flanked by the transframe region. Substituting PR20 cluster 3 into TFR-PR (TFR-PR(PR20-3)) requires the presence of PR20 cluster 1 and/or 2 for autoprocessing. In accordance, substituting PR clusters 1 and 2 into TFR-PR20 affects the rate of autoprocessing more drastically (>300-fold) compared to that of TFR-PR(PR20-3) because of the cumulative effect of eight noncluster mutations present in TFR-PR20(PR-12). Overall, these studies imply that drug resistance involves a complex synchronized selection of mutations modulating all of the properties mentioned above governing PR regulation and function.

Enhanced Stability of Monomer Fold Correlates with Extreme Drug Resistance of HIV-1 Protease

During treatment, mutations in HIV-1 protease (PR) are selected rapidly that confer resistance by decreasing affinity to clinical protease inhibitors (PIs). As these unique drug resistance mutations can compromise the fitness of the virus to replicate, mutations that restore conformational stability and activity while retaining drug resistance are selected on further evolution. Here we identify several compensating mechanisms by which an extreme drug-resistant mutant bearing 20 mutations (PR20) with >5-fold increased Kd and >4000-fold decreased affinity to the PI darunavir functions. (1) PR20 cleaves, albeit poorly, Gag polyprotein substrates essential for viral maturation. (2) PR20 dimer, which exhibits distinctly enhanced thermal stability, has highly attenuated autoproteolysis, thus likely prolonging its lifetime in vivo. (3) The enhanced stability of PR20 results from stabilization of the monomer fold. Both monomeric PR20(T26A) and dimeric PR20 exhibit Tm values 6-7.5 °C higher than those for their PR counterparts. Two specific mutations in PR20, L33F and L63P at sites of autoproteolysis, increase the Tm of monomeric PR(T26A) by ~8 °C, similar to PR20(T26A). However, without other compensatory mutations as seen in PR20, L33F and L63P substitutions, together, neither restrict autoproteolysis nor significantly reduce binding affinity to darunavir. To determine whether dimer stability contributes to binding affinity for inhibitors, we examined single-chain dimers of PR and PR(D25N) in which the corresponding identical monomer units were covalently linked by GGSSG sequence. Linking of the subunits did not appreciably change the ΔTm on inhibitor binding; thus stabilization by tethering appears to have little direct effect on enhancing inhibitor affinity.

Mechanism of dissociative inhibition of HIV protease and its autoprocessing from a precursor.

Dimerization is indispensible for release of the human immunodeficiency virus protease (PR) from its precursor (Gag-Pol) and ensuing mature-like catalytic activity that is crucial for virus maturation. We show that a single-chain Fv fragment (scFv) of a previously reported monoclonal antibody (mAb1696), which recognizes the N-terminus of PR, dissociates a dimeric mature D25N PR mutant with an enhanced dimer dissociation constant (K(d)) in the sub-micromolar range to form predominantly a monomer-scFv complex at a 1:1 ratio, along with small (5-10%) amounts of a dimer-scFv complex. Enzyme kinetics indicate a mixed mechanism of inhibition of the wild-type PR, which exhibits a K(d)<10nM, with effects both on K(m) and k(cat) at an scFv-to-PR ratio of 10:1. ScFv binds to the N-terminal peptide P(1)QITLW(6) of PR and to PR monomers with dissociation constants of ≤30 nM and ~100 nM, respectively. Consistent with an ~400-fold increase in the dissociation of the antibody (K(Ab)) on even addition of an acetyl group to P(1) of the peptide, the antibody fails to inhibit N-terminal autoprocessing of the PR from a model precursor (at ~5 μM). However, subsequent to this cleavage, it sequesters the PR, thus blocking autoprocessing at its C-terminus. A second monoclonal antibody [PRM1 (human monoclonal antibody to PR)], which recognizes part of the flap region (residues 41-47) of the mature PR and its precursor, does not inhibit autoprocessing and ensuing catalytic activity. However, its failure to recognize drug-resistant clinical mutants of PR may be beneficial to monitor the selection of mutations in this region under drug pressure.

Binding of HIV-1 gp41-Directed Neutralizing and Non- Neutralizing Fragment Antibody Binding Domain (Fab) and Single Chain Variable Fragment (ScFv) Antibodies to the Ectodomain of gp41 in the Pre-Hairpin and Six-Helix Bundle Conformations

We previously reported a series of antibodies, in fragment antigen binding domain (Fab) formats, selected from a human non-immune phage library, directed against the internal trimeric coiled-coil of the N-heptad repeat (N-HR) of HIV-1 gp41. Broadly neutralizing antibodies from that series bind to both the fully exposed N-HR trimer, representing the pre-hairpin intermediate state of gp41, and to partially-exposed N-HR helices within the context of the gp41 six-helix bundle. While the affinities of the Fabs for pre-hairpin intermediate mimetics vary by only 2 to 20-fold between neutralizing and non-neutralizing antibodies, differences in inhibition of viral entry exceed three orders of magnitude. Here we compare the binding of neutralizing (8066) and non-neutralizing (8062) antibodies, differing in only four positions within the CDR-H2 binding loop, in Fab and single chain variable fragment (ScFv) formats, to several pre-hairpin intermediate and six-helix bundle constructs of gp41. Residues 56 and 58 of the mini-antibodies are shown to be crucial for neutralization activity. There is a large differential (≥150-fold) in binding affinity between neutralizing and non-neutralizing antibodies to the six-helix bundle of gp41 and binding to the six-helix bundle does not involve displacement of the outer C-terminal helices of the bundle. The binding stoichiometry is one six-helix bundle to one Fab or three ScFvs. We postulate that neutralization by the 8066 antibody is achieved by binding to a continuum of states along the fusion pathway from the pre-hairpin intermediate all the way to the formation of the six-helix bundle, but prior to irreversible fusion between viral and cellular membranes.

Insights into the Conformation of the Membrane Proximal Regions Critical to the Trimerization of the HIV-1 gp41 Ectodomain Bound to Dodecyl Phosphocholine Micelles.

The transitioning of the ectodomain of gp41 from a pre-hairpin to a six-helix bundle conformation is a crucial aspect of virus-cell fusion. To gain insight into the intermediary steps of the fusion process we have studied the pH and dodecyl phosphocholine (DPC) micelle dependent trimer association of gp41 by systematic deletion analysis of an optimized construct termed 17–172 (residues 528 to 683 of Env) that spans the fusion peptide proximal region (FPPR) to the membrane proximal external region (MPER) of gp41, by sedimentation velocity and double electron-electron resonance (DEER) EPR spectroscopy. Trimerization at pH 7 requires the presence of both the FPPR and MPER regions. However, at pH 4, the protein completely dissociates to monomers. DEER measurements reveal a partial fraying of the C-terminal MPER residues in the 17–172 trimer while the other regions, including the FPPR, remain compact. In accordance, truncating nine C-terminal MPER residues (675–683) in the 17–172 construct does not shift the trimer-monomer equilibrium significantly. Thus, in the context of the gp41 ectodomain spanning residues 17–172, trimerization is clearly dependent on FPPR and MPER regions even when the terminal residues of MPER unravel. The antibody Z13e1, which spans both the 2F5 and 4E10 epitopes in MPER, binds to 17–172 with a Kd of 1 ± 0.12 μM. Accordingly, individual antibodies 2F5 and 4E10 also recognize the 17–172 trimer/DPC complex. We propose that binding of the C-terminal residues of MPER to the surface of the DPC micelles models a correct positioning of the trimeric transmembrane domain anchored in the viral membrane.